CAMP factor of Streptococcus uberis

ABSTRACT

The CAMP factor gene of  Streptococcus uberis  ( S. uberis ) is described, as well as the recombinant production of CAMP factor therefrom. Also disclosed are chimeric CAMP factor constructs, including CAMP factor epitopes from more than one bacterial species. The CAMP factors and chimeras including the same can be used in immunogenic compositions for the prevention and treatment of bacterial infections.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/234,733, filed Jan. 21, 1999, which is a divisional of U.S.patent application Ser. No. 08/658,277, filed Jun. 5, 1996, now issuedas U.S. Pat. No. 5,863,543, from which applications priority is claimedunder 35 USC §120, which is related to provisional patent applicationSer. No. 60/000,083, filed Jun. 8, 1995, from which application priorityis claimed under 35 USC §119(e)(1), and which applications areincorporated herein by reference in their entireties.

TECHNICAL FIELD

The present invention relates generally to bacterial antigens. Moreparticularly, the present invention pertains to the recombinantproduction of CAMP factor from Streptococcus uberis (S. uberis) and theuse of CAMP factors in vaccine compositions. The present invention alsopertains to the production and use of chimeric CAMP factor constructs,comprising epitopes of CAMP factors from more than one bacterialspecies.

BACKGROUND

S. uberis is an important cause of mastitis in dairy cattle and isresponsible for about 20% of all clinical cases of mastitis (Bramley, A.J. and Dodd, F. H. (1984) J. Dairy Res. 51:481-512; Bramley, A. J.(1987) Animal Health Nutrition 42:12-16; Watts, J. L. (1988) J. DairySci. 71:1616-1624). Since antimicrobial treatment is generallyineffective in treating S. uberis mastitis, the development of controlmeasures must be based on an understanding of virulence factors andprotective antigens involved in invasion and protection of the mammarygland (Collins et al. (1988) J. Dairy Res. 55:25-32; Leigh et al. (1990)Res. Vet. Sci. 49: 85-87; Marshall et al. (1986) J. Dairy Res. 53:507-514).

It is known that some S. uberis strains can produce hyaluronic acidcapsule (Hill, A. W. (1988) Res. Vet. Sci. 45:400-404), hyaluronidase(Schaufuss et al. (1989) Zentralbl. Bakteriol. Ser. A 271:46-53), R-likeprotein (Groschup, M. H. and Timoney, J. F. (1993) Res. Vet. Sci.54:124-126), and a cohemolysin, the CAMP factor, also known as UBERISfactor (Skalka, B. and Smola, J. (1981) Zentralbl. Bakteriol. Ser. A249:190-194). However, very little is known of their roles inpathogenicity.

The effect of CAMP factor was first described by Christie et al. in 1944(Christie et al. (1944) Aus. J. Exp. Biol. Med. Sci. 22:197-200). Theseauthors found that group B streptococci (GBS), such as S. agalactiae,produced a distinct zone of complete hemolysis when grown near thediffusion zone of the Staphylococcus aureus beta-toxin,sphingomyelinase. This phenomenon was called CAMP reaction and thecompound for this reaction was characterized as the CAMP factor, anextracellular protein with a molecular weight of 23,500 (Bernheimer etal. (1979) Infect. Immun. 23:838-844). The CAMP factor was subsequentlypurified from S. agalactiae and characterized as a 25,000 Da proteinwith a pI of 8.9 (Jürgens et al. (1985) J. Chrom. 348:363-370). Theamino acid sequence of S. agalactiae CAMP factor was determined byRühlmann et al. (Rühlmann et al. (1988) FEBS Lett 235:262-266).

The mechanism of the CAMP reaction has been described. See, e.g.,Bernheimer et al. (1979) Infect. Immun. 23:838-844; Sterzik et al.“Interaction of the CAMP factor from S. agalactiae with artificialmembranes.” In: Alouf et al., eds. Bacterial protein toxins, London:Academic Press Inc, 1984; 195-196; Sterzik et al. (1985) Zentralbl.Bakteriol. Mikrobiol. Hyg. Abt. 1 Suppl. 15:101-108; Fehrenbach et al.“Role of CAMP-factor (protein B) for virulence.” In: Fehrenbach et al.,eds. Bacterial protein toxins, Stuttgart: Gustav Fischer Verlag, 1988;351-357; Fehrenbach et al. “Interaction of amphiphilic bacterialpolypeptides with artificial membranes.” In: Alouf et al., eds.Bacterial protein toxins, London: Academic Press Inc., 1984:317-324.

CAMP factor has lytic action on a variety of target cells includingsheep and bovine erythrocytes, as well as on artificial membranes inwhich membrane phospholipids and sphingomyelin have been hydrolyzed byphospholipase or sphingomyelinase.

The role of CAMP factor in pathogenicity is unclear. A partiallypurified CAMP factor from S. agalactiae has been shown to be lethal torabbits when injected intravenously (Skalka, B. and Smola, J. (1981)Zentralbl. Bakteriol. Ser. A 249:190-194). Furthermore, intraperitonealinjection of purified CAMP factor into mice has been shown tosignificantly raise the pathogenicity of a sublethal dose of group Bstreptococci (Fehrenbach et al. “Role of CAMP-factor (protein B) forvirulence.” In: Fehrenbach et al., eds. Bacterial protein toxins,Stuttgart: Gustav Fischer Verlag, 1988; 351-357). Additionally, likeprotein A of S. aureus, GBS CAMP factor can bind the Fc sites ofimmunoglobulins and has therefore been designated protein B (Jürgens etal. (1987) J. Exp. Med. 165:720-732).

In addition to GBS and S. uberis, other bacteria, including Listeriamonocytogenes and Listeria seeligeri (Rocourt, J. and Grimont, P. A. D.(1983) Int. J. Syst. Bacteriol. 33:866-869) Aeromonas sp. (Figura, N.and Guglielmetti, P. (1987) J. Clin. Microbiol. 25:1341-1342),Rhodococcus equi (Fraser, G. (1961) Nature 189:246), and certain Vibriospp. (Kohler, W. (1988) Zentralbl. Bakteriol. Mikrobiol. Hyg. Ser. A270:35-40) produce reactions similar to the CAMP effect.

The CAMP factor genes of GBS and A. pleuropneumoniae have been clonedand expressed in Escherichia coli (Schneewind et al. (1988) Infect.Immun. 56:2174-2179; Frey et al. (1989) Infect. Immun. 57:2050-2056).Additionally, the gene encoding the CAMP factor from a group Astreptococci (GAS) strain, S. pyogenes, has also been isolated (Gase etal. (1999) Infect. Immun. 67:4725-4731). The CAMP protein products wereof similar size and possessed homology to the CAMP proteins of S.agalactiae and S. uberis. Antibodies raised against the cloned CAMPprotein of A. pleuropneumoniae neutralized the CAMP reaction mediated bythe E. coli strain containing the cloned CAMP gene as well as that of A.pleuropneumoniae, and also cross-reacted with the S. agalactiae CAMPfactor. In the GAS strains, the distribution of the cfa (CAMP) gene wasanalyzed. This gene was widely spread among GAS: 82 of 100 clinical GASisolates produced a positive CAMP reaction. Of the CAMP-negativestrains, 17 of the 18 GAS strains contained the cfa gene. Additionally,CAMP activity was detected in streptococci from serogroups C, M, P, R,and U (Gase et al. (1999) Infect. Immun. 67:4725-4731).

However, prior to the present inventors' discovery, the CAMP factor geneof S. uberis had not been cloned. Furthermore, the protective capabilityof CAMP factor had not been previously studied. Additionally, theproduction and use of chimeric CAMP factor constructs, containingepitopes derived from CAMP factors from more than one microbe, has notpreviously been described.

DISCLOSURE OF THE INVENTION

The present invention is based on the discovery of the CAMP factor geneof S. uberis, as well as the discovery that the CAMP factor, andchimeric constructs including multiple CAMP factor epitopes, is able toprotect vertebrate subjects from infection. The CAMP factor, activeimmunogenic fragments thereof, active analogs thereof, or chimericproteins including multiple CAMP factors from more than one organism,can be used, either alone or in combination with other antigens, innovel subunit vaccines to provide protection from bacterial infection invertebrate subjects, or as diagnostic reagents.

Accordingly, in one embodiment, the subject invention is directed to animmunogenic polypeptide comprising one or more CAMP factor epitopes frommore than one bacterial species. In certain embodiments, the one or moreCAMP factor epitopes are separated by a spacer consisting of 1-20 aminoacids. Moreover, the one or more CAMP factor epitopes may be from morethan one bacterial species of the genus Streptococcus, such as oneselected from the group consisting of S. uberis, S. agalactiae and S.pyogenes.

In additional embodiments, at least one of the CAMP factor epitopes isfrom the CAMP factor N-terminal variable region corresponding to theregion defined by amino acids 1-90 of FIG. 2. In certain embodiments,the epitope is interposed within a CAMP factor protein having at least80% sequence identity to the CAMP factor protein of FIG. 2 or FIG. 3,and the CAMP factor protein is from a different streptococcal speciesthan the CAMP factor epitope from the N-terminal variable region. Inparticular embodiments, the CAMP factor epitope from the N-terminalvariable region comprises a sequence of amino acids having at least 80%sequence identity to the sequence of amino acids depicted at positions31-87 of FIG. 3 and the CAMP factor protein has at least 80% sequenceidentity to the CAMP factor protein of FIG. 2.

In other embodiments, the immunogenic polypeptide comprises a sequenceof amino acids having at least 80% sequence identity to the contiguoussequence of amino acids depicted at positions 27-314 of FIG. 8, with orwithout a signal sequence. In yet another embodiment, the immunogenicpolypeptide comprises the amino acid sequence depicted in FIG. 8.

In another embodiment, the invention is directed to immunogeniccompositions comprising an immunogenic protein as detailed above, and apharmaceutically acceptable. The composition can additionally comprisean adjuvant.

In further embodiments, the invention is directed to a method ofproducing an immunogenic composition comprising the steps of:

-   -   (1) providing an immunogenic polypeptide as described above; and    -   (2) combining said polypeptide with a pharmaceutically        acceptable vehicle.

In additional embodiments, the invention is directed to a method oftreating or preventing a bacterial infection in a vertebrate subjectcomprising administering to the subject a therapeutically effectiveamount of an immunogenic composition as detailed above. In certainembodiments, the infection is a streptococcal infection and causesmastitis.

In yet further embodiments, the invention pertains to antibodiesdirected against the immunogenic polypeptides described above. Theantibodies can be polyclonal or monoclonal.

In additional embodiments, the invention is directed to a polynucleotidecomprising a coding sequence encoding an immunogenic polypeptide asdescribed above, a recombinant vector comprising the polynucleotide andat least one control element operably linked to polynucleotide, wherebythe coding sequence can be transcribed and translated in a host cell, ahost cell comprising the recombinant vector, and a method for producingan immunogenic polypeptide, the method comprising culturing a populationof host cells as described above under conditions for producing saidpolypeptide.

In other embodiments, the invention is directed to a method of detectingStreptococcus antibodies in a biological sample, comprising:

(a) reacting the biological sample with the immunogenic polypeptide asdescribed above, under conditions which allow Streptococcus antibodies,when present in the biological sample, to bind to the polypeptide toform an antibody/antigen complex; and

(b) detecting the presence or absence of the complex, and therebydetecting the presence or absence of Streptococcus antibodies in thesample.

In yet other embodiments, the invention is directed to animmunodiagnostic test kit for detecting Streptococcus infection, thetest kit comprising an immunogenic polypeptide as described above andinstructions for conducting the immunodiagnostic test.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts restriction enzyme maps of recombinant plasmid pJLD21 andsubclones thereof, designated pJLD21-1 and pJLD21-2. Lines indicate S.uberis insert DNA, while boxes represent the multiple cloning sites ofvector pTZ18R. The CAMP activities of recombinant plasmid pJLD21 and itsderived subclones are indicated on the right (+, CAMP reaction positive;−, CAMP reaction negative). The small horizontal arrows represent startpoints and directions of sequencing experiments. The probe fragment usedfor Southern blot experiments is indicated by the large arrow. The barat the bottom indicates the location of the open reading frame of CAMPfactor gene of S. uberis (cfu).

FIG. 2 (SEQ ID NOS:1 and 2) shows the nucleotide sequence and thecorresponding amino acid sequence of the S. uberis CAMP factor. Thenucleotide positions are indicated on the left while amino acidpositions are denoted on the right. A signal peptide occurs at aminoacids 1-30 of the figure and the mature peptide is represented by aminoacid positions 31-258.

FIG. 3 shows (SEQ ID NOS:3 and 4) shows the nucleotide sequence and thecorresponding amino acid sequence of the S. agalactiae CAMP factor. Thenucleotide positions are indicated on the left while amino acidpositions are denoted on the right. A signal peptide occurs at aminoacids 1-29 of the figure and mature peptide is represented by amino acidpositions 30-255.

FIG. 4 shows the homology between S. agalactiae (SEQ ID NO:4) and S.uberis (SEQ ID NO:2) CAMP proteins. A consensus sequence (SEQ ID NO:5)is also shown. Alignment of the published sequences of the S. agalactiae(AgalCAMP) and S. uberis (UberCAMP) CAMP proteins were generated withPileUp, and displayed with Pretty software (a component of the GCGWisconsin Package, version 10, provided by the SeqWeb sequence analysispackage, version 1.1, of the Canadian Bioinformatics Resource). Theconsensus sequence is shown below the aligned proteins. The residues arenumbered on top. The wavy lines indicate spaces added by the algorithmto correct for different size-proteins. The dots depict gaps between thealigned sequences. The dashes in the consensus sequences representnon-identical residues. The parameters used for the alignment were:Comparison table: blosum62; Gap weight: 8 and Gap length weight: 2. Thestart of the mature sequence is indicated with an arrow. The alignedsequences show approximately 69% sequence similarity and 63% sequenceidentity. Amino acid residues 1-30 of the S. uberis sequence shown inthe figure represent the signal sequence while amino acid residues 1-29of the S. agalactiae sequence represent the signal sequence.

FIG. 5 depicts the construction of pGH-CAMP. The CAMP reactions for eachclone from which the plasmid was derived are shown to the right of thefigure; + indicates a positive reaction and − a negative reaction. Openboxes indicate pTZ18R and hatched boxes indicate pGH433.

FIG. 6 shows the putative signal sequence of S. agalactiae CAMP factor(FIG. 6A; SEQ ID NO:6) and S. uberis CAMP factor (FIG. 6B; SEQ ID NO:7).The predicted Signal Peptidase I cleavage sites (Indicated by ^) of theS. agalactiae and S. uberis CAMP proteins are shown. Also shown are theprobability score and the length of the signal peptide for each of thetwo CAMP proteins. The sequences were analyzed by the SPScan algorithmof the GCG software package.

FIGS. 7A-7E show the plasmids used to construct the CAMP-3 chimera. Theintermediate and final plasmids are shown. The restriction enzymes,location, and orientation of relevant genes are shown for each plasmid.The construction of these plasmids is explained in the experimentalsection.

FIG. 8 depicts the nucleotide and amino acid sequences of theLipoF:CAMP3 chimera (SEQ ID NOS:8 and 9). The nucleotide and proteinsequence of the CAMP-3 chimera fused to the LipoF signal sequence isshown. The nucleotides are numbered on the left of the figure, while theamino acid residues are numbered on the right. The underlined sequencerepresents the LipoF signal sequence. The spacer amino acids occur atpositions 87, 145 and 146.

FIG. 9 shows the geometric mean SCC (plus 1 standard deviation) inquarters challenged with S. uberis in groups of cows as described in theexamples, for 7 days following challenge. Data sets correspond to day 0(the far left bar), day 1 (the second bar from the left), day 2 (thethird bar from the left), day 3 (the fourth bar from the left), day 4(the fifth bar from the left), day 5 (the sixth bar from the left), day6 (the seventh bar from the left), and day 7 (the eighth bar from theleft). Despite the appearance of the figure, the SCC for S. dysgalactiae(6×His)GapC vaccinated animals on days 7 and 8, and CAMP-3 vaccinatedanimals on days 4 and 5 post-challenge were not statisticallysignificantly different from those of the control.

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,virology, recombinant DNA technology, and immunology, which are withinthe skill of the art. Such techniques are explained filly in theliterature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II(D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed.1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.1984); Animal Cell Culture (R. K. Freshney ed. 1986); Immobilized Cellsand Enzymes (IRL press, 1986); Perbal, B., A Practical Guide toMolecular Cloning (1984); the series, Methods In Enzymology (S. Colowickand N. Kaplan eds., Academic Press, Inc.); and Handbook of ExperimentalImmunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986,Blackwell Scientific Publications).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

The following amino acid abbreviations are used throughout the text:

Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (N) Aspartic acid:Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic acid: Glu (E)Glycine: Gly (G) Histidine: His (H) Isoleucine: Ile (I) Leucine: Leu (L)Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Proline: Pro(P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr(Y) Valine: Val (V)A. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a CAMP factor” includes a mixture of two or more CAMPfactors, and the like.

The term “CAMP factor” or a nucleotide sequence encoding the same,intends a protein or a nucleotide sequence, respectively, which isderived from a CAMP factor gene found in a variety of bacterial species,including, without limitation Streptococcus uberis, group B streptococci(GBS) such as S. agalactiae (Jürgens et al. (1985) J. Chromatogr.348:363-370; Rühlmann et al. (1988) FEBS Lett 235:262-266; Schneewind etal. (1988) Infect. Immun. 56:2174-2179), A. pleuropneumoniae (Frey etal. (1989) Infect. Immun. 57:2050-2056), group A streptococci (GAS),such as S. pyogenes (Gase et al. (1999) Infect. Immun. 67:4725-4731),and streptococci from serogroups C, M, P, R, and U, Listeriamonocytogenes and Listeria seeligeri (Rocourt, J. and Grimont, P. A. D.(1983) Int. J. Syst. Bacteriol. 33:866-869), Aeromonas sp. (Figura, N.and Guglielmetti, P. (1987) J. Clin. Microbiol. 25:1341-1342),Rhodococcus equi (Fraser, G. (1961) Nature 189:246), and certain Vibriospp. (Kohler, W. (1988) Zentralbl. Bakteriol. Mikrobiol. Hyg. Ser. A270:35-40).

A representative CAMP factor gene, derived from S. uberis, is found inplasmid pJLD21 (ATCC Accession No. 69837). The nucleotide sequence andcorresponding amino acid sequence for the S. uberis CAMP factor isdepicted in FIG. 2 (SEQ ID NOS:1 and 2). FIG. 3 (SEQ ID NOS:3 and 4)shows a nucleotide sequence and corresponding amino acid sequence forthe S. agalactiae CAMP factor. The sequences for other CAMP factors areknown and described in the art as detailed above.

However, a CAMP factor, as defined herein is not limited to the depictedand described sequences as subtypes of each of these bacterial speciesare known and variations in CAMP proteins will occur between them.Moreover, the derived protein or nucleotide sequences need not bephysically derived from the gene described above, but may be generatedin any manner, including for example, chemical synthesis, isolation(e.g., from an organism that produces the CAMP factor) or by recombinantproduction, based on the information provided herein.

The term also includes proteins possessing, as well as lacking, a signalsequence, if such is present. As shown in FIGS. 2, 3 and 4, S. uberisand S. agalactiae both include signal sequences occurring at amino acidpositions 1-30 and 1-29, respectively. In general, the term “mature”CAMP factor refers to a CAMP factor lacking the signal sequence. Thus,the term “mature” CAMP factor with reference to the sequences depictedin FIGS. 2 and 3, refers to the amino acid sequence shown at positions31-258 of FIG. 2 and positions 30-255 of FIG. 3. The term CAMP factoralso refers to proteins in neutral form or in the form of basic or acidaddition salts depending on the mode of preparation. Such acid additionsalts may involve free amino groups and basic salts may be formed withfree carboxyls. Pharmaceutically acceptable basic and acid additionsalts are discussed further below. In addition, the proteins may bemodified by combination with other biological materials such as lipids(both those occurring naturally with the molecule or other lipids thatdo not destroy immunological activity) and saccharides, or by side chainmodification, such as acetylation of amino groups, phosphorylation ofhydroxyl side chains, oxidation of sulfhydryl groups, glycosylation ofamino acid residues, as well as other modifications of the encodedprimary sequence.

The term “streptococcal CAMP factor” intends a CAMP factor, as definedabove, derived from a streptococcal species that produces the same,including, without limitation, S. uberis, GBS such as S. agalactiae,GAS, such as S. pyogenes, and streptococci from serogroups C, M, P, R,and U. For example, an “S. uberis CAMP factor” is a CAMP factor asdefined above, derived from S. uberis. An “S. agalactiae CAMP factor” isa CAMP factor as defined above, derived from S. agalactiae.

The terms “variant” and “mutein” of a CAMP factor protein refer tobiologically active derivatives of a CAMP factor, as defined above, orfragments of such derivatives, that retain immunological activity asdefined herein. The term “mutein” refers to peptides having one or morepeptide mimics (“peptoids”), such as those described in InternationalPublication No. WO 91/04282. Preferably, the variant or mutein has atleast the same activity as the native molecule. Methods for makingpolypeptide variants and muteins are known in the art and are describedfurther below.

In general, the term “variant” refers to compounds having a nativepolypeptide sequence and structure with one or more amino acidadditions, substitutions (generally conservative in nature) and/ordeletions, relative to the native molecule, so long as the modificationsdo not destroy activity. Thus, a “variant” of a CAMP factor proteinincludes a protein with amino acid sequences substantially homologous(as defined below) to contiguous amino acid sequences encoded by theCAMP factor genes, which display immunological activity. Particularlypreferred substitutions will generally be conservative in nature, i.e.,those substitutions that take place within a family of amino acids. Forexample, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cystine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, or viceversa; an aspartate with a glutamate or vice versa; a threonine with aserine or vice versa; or a similar conservative replacement of an aminoacid with a structurally related amino acid, will not have a majoreffect on the biological activity. Proteins having substantially thesame amino acid sequence as the reference molecule, but possessing minoramino acid substitutions that do not substantially affect theimmunogenicity of the protein, are therefore within the definition ofthe reference polypeptide.

Other substitutions include substitutions of naturally occurring aminoacids with amino acid analogs. Such amino acid analogs are well knownand include, but are not limited to, 2-aminoadipic acid, 3-aminoadipicacid, beta-alanine, beta-aminopropionic acid, 2-aminobutyric acid,4-aminobutyric acid, piperidinic acid, 6-aminocaproic acid,2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid,2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine,2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine,N-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline,4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine,sarcosine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline,norvaline, norleucine and ornithine.

For example, the polypeptide of interest may include up to about 5-10conservative or non-conservative amino acid substitutions, or even up toabout 15-25 or 20-50 conservative or non-conservative amino acidsubstitutions, or any integer between these values, so long as thedesired function of the molecule remains intact.

By “fragment” is intended a polypeptide or polynucleotide consisting ofonly a part of the intact polypeptide sequence and structure, or thenucleotide sequence and structure, of the reference gene. Thepolypeptide fragment can include a C-terminal deletion and/or N-terminaldeletion of the native polypeptide, or can be derived from an internalportion of the molecule. Similarly, a polynucleotide fragment caninclude a 3′ and/or a 5′ deletion of the native polynucleotide, or canbe derived from an internal portion of the molecule. A polypeptide“fragment” of a CAMP factor will generally include at least about 2-5contiguous amino acid residues, preferably at least about 10 contiguousamino acid residues of the full-length molecule, preferably at leastabout 15-25 contiguous amino acid residues of the full-length molecule,and most preferably at least about 20-50 or more contiguous amino acidresidues of full-length molecule, or any integer between 2 amino acidsand the full-length sequence, provided that the fragment in questionretains desired activity.

A nucleotide fragment of the gene of interest generally includes atleast about 8 contiguous base pairs, more preferably at least about10-20 contiguous base pairs, and most preferably at least about 25-50,or more, contiguous base pairs of the gene, or any integers betweenthese values. Such fragments are useful as probes and in diagnosticmethods, discussed more fully below.

By “mastitis” is meant an inflammation of the mammary gland in mammals,including in cows, ewes, goats, sows, mares, and the like, caused byvarious bacteria that produce CAMP factors, described more fully below.The infection manifests itself by the infiltration of phagocytic cellsin the gland. Generally, 4 clinical types of mastitis are recognized:(1) peracute, associated with swelling, heat, pain, and abnormalsecretion in the gland and accompanied by fever and other signs ofsystemic disturbance, such as marked depression, rapid weak pulse,sunken eyes, weakness and complete anorexia; (2) acute, with changes inthe gland similar to those above but where fever, anorexia anddepression are slight to moderate; (3) subacute, where no systemicchanges are displayed and the changes in the gland and its secretion areless marked: and (4) subclinical, where the inflammatory reaction isdetectable only by standard tests for mastitis.

Standard tests for the detection of mastitis include but are not limitedto, the California Mastitis Test, the Wisconsin Mastitis Test, theNagase test, the electronic cell count and somatic cell counts used todetect a persistently high white blood cell content in milk. In general,a somatic cell count of about 300,000 to about 500,000 cells per ml orhigher, in milk will indicate the presence of infection. Thus, a vaccineis considered effective in the treatment and/or prevention of mastitiswhen, for example, the somatic cell count in milk is retained belowabout 500,000 cells per ml. For a discussion of mastitis and thediagnosis thereof, see, e.g., The Merck Veterinary Manual. A Handbook ofDiagnosis, Therapy, and Disease Prevention and Control for theVeterinarian, Merck and Co., Rahway, N.J., 1991.

The term “epitope” refers to the site on an antigen or hapten to whichspecific B cells and T cells respond. The term is also usedinterchangeably with “antigenic determinant” or “antigenic determinantsite.” Antibodies that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen.

An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to the composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells and/or γδ T cells,directed specifically to an antigen or antigens included in thecomposition or vaccine of interest. Preferably, the host will display aprotective immunological response to the CAMP factor in question, e.g.,the host will be protected from subsequent infection by the pathogen andsuch protection will be demonstrated by either a reduction or lack ofsymptoms normally displayed by an infected host or a quicker recoverytime.

The terms “immunogenic” protein or polypeptide refer to an amino acidsequence which elicits an immunological response as described above. An“immunogenic” protein or polypeptide, as used herein, includes thefull-length sequence of the CAMP factor in question, including theprecursor and mature forms, analogs thereof, or immunogenic fragmentsthereof.

By “immunogenic fragment” is meant a fragment of a CAMP factor whichincludes one or more epitopes and thus elicits the immunologicalresponse described above. Such fragments can be identified using anynumber of epitope mapping techniques, well known in the art. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66(Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example,linear epitopes may be determined by e.g., concurrently synthesizinglarge numbers of peptides on solid supports, the peptides correspondingto portions of the protein molecule, and reacting the peptides withantibodies while the peptides are still attached to the supports. Suchtechniques are known in the art and described in, e.g., U.S. Pat. No.4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002;Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated hereinby reference in their entireties. Similarly, conformational epitopes arereadily identified by determining spatial conformation of amino acidssuch as by, e.g., x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols, supra.Antigenic regions of proteins can also be identified using standardantigenicity and hydropathy plots, such as those calculated using, e.g.,the Omiga version 1.0 software program available from the OxfordMolecular Group. This computer program employs the Hopp/Woods method,Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78:3824-3828 fordetermining antigenicity profiles, and the Kyte-Doolittle technique,Kyte et al., J. Mol. Biol. (1982) 157:105-132 for hydropathy plots.

Immunogenic fragments, for purposes of the present invention, willusually be at least about 2 amino acids in length, more preferably about5 amino acids in length, and most preferably at least about 10 to 15amino acids in length. There is no critical upper limit to the length ofthe fragment, which could comprise nearly the full-length of the proteinsequence, or even a fusion protein comprising two or more epitopes ofthe CAMP factor or factors in question.

An “immunogenic composition” is a composition that comprises anantigenic molecule where administration of the composition to a subjectresults in the development in the subject of a humoral and/or a cellularimmune response to the antigenic molecule of interest.

By “subunit vaccine composition” is meant a composition containing atleast one immunogenic polypeptide, but not all antigens, derived from orhomologous to an antigen from a pathogen of interest. Such a compositionis substantially free of intact pathogen cells or particles, or thelysate of such cells or particles. Thus, a “subunit vaccine composition”is prepared from at least partially purified (preferably substantiallypurified) immunogenic polypeptides from the pathogen, or recombinantanalogs thereof. A subunit vaccine composition can comprise the subunitantigen or antigens of interest substantially free of other antigens orpolypeptides from the pathogen.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual in a formulationor composition without causing any undesirable biological effects orinteracting in a deleterious manner with any of the components of thecomposition in which it is contained.

The term “multiple epitope” protein or polypeptide specifies a sequenceof amino acids comprising an epitope as defined herein, which containsat least one epitope repeated two or more times within a linearmolecule. The repeating sequence need not be directly connected toitself, is not repeated in nature in the same manner and, further, maybe present within a larger sequence which includes other amino acidsthat are not repeated. For the purposes of this invention, the epitopesequence may either be an exact copy of a wild-type epitope sequence, ora sequence which is “functionally equivalent” as defined herein.

A “fusion” or “chimeric” protein or polypeptide is one in which aminoacid sequences from more than one source are joined. Such molecules maybe produced synthetically or recombinantly, as described further herein.A representative chimeric protein comprising the mature CAMP protein ofS. uberis as a backbone, and an N-terminal peptide from the S.agalactiae CAMP protein from a region with low homology to the S. uberisCAMP product, is shown at amino acid positions 27-314 of FIG. 8. Spacersequences occur at positions 87, 145 and 146. The CAMP chimera depictedin the Figure was fused to the LipoF signal sequence, represented bypositions 1-26 of FIG. 8.

An “isolated” nucleic acid molecule is a nucleic acid molecule separateand discrete from the whole organism with which the molecule is found innature; or a nucleic acid molecule devoid, in whole or part, ofsequences normally associated with it in nature; or a sequence, as itexists in nature, but having heterologous sequences (as defined below)in association therewith.

“Recombinant” polypeptides refer to polypeptides produced by recombinantDNA techniques; i.e., produced from cells transformed by an exogenousDNA construct encoding the desired polypeptide. “Synthetic” polypeptidesare those prepared by chemical synthesis.

A “vector” is a replicon, such as a plasmid, phage, or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A DNA “coding sequence” or a “nucleotide sequence encoding” a particularprotein, is a DNA sequence which is transcribed and translated into apolypeptide in vitro or in vivo when placed under the control ofappropriate regulatory elements. The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, procaryotic sequences, cDNA fromeucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian)DNA, and even synthetic DNA sequences. A transcription terminationsequence will usually be located 3′ to the coding sequence.

DNA “control elements” refers collectively to promoters, ribosomebinding sites, polyadenylation signals, transcription terminationsequences, upstream regulatory domains, enhancers, and the like, whichcollectively provide for the transcription and translation of a codingsequence in a host cell. Not all of these control sequences need alwaysbe present in a recombinant vector so long as the desired gene iscapable of being transcribed and translated.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter and the coding sequence and the promoter canstill be considered “operably linked” to the coding sequence.

Similarly, a coding sequence is “operably linked to” another codingsequence (i.e., in the case of a chimeric protein) when RNA polymerasewill transcribe the two coding sequences into mRNA, which is thentranslated into the polypeptides encoded by the two coding sequences.The coding sequences need not be contiguous to one another so long asthe transcribed sequence is ultimately processed to produce the desiredprotein.

A control element, such as a promoter, “directs the transcription” of acoding sequence in a cell when RNA polymerase will bind the promoter andtranscribe the coding sequence into mRNA, which is then translated intothe polypeptide encoded by the coding sequence.

A “host cell” is a cell which has been transformed, or is capable oftransformation, by an exogenous nucleic acid molecule.

A cell has been “transformed” by exogenous DNA when such exogenous DNAhas been introduced inside the cell membrane. Exogenous DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In procaryotes and yeasts, for example, theexogenous DNA may be maintained on an episomal element, such as aplasmid. With respect to eucaryotic cells, a stably transformed cell isone in which the exogenous DNA has become integrated into the chromosomeso that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeucaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the exogenous DNA.

“Homology” refers to the percent similarity between two polynucleotideor two polypeptide moieties. Two DNA, or two polypeptide sequences are“substantially homologous” to each other when the sequences exhibit atleast about 80%-85%, preferably at least about 90%, and most preferablyat least about 95%-98% sequence similarity or identity over a definedlength of the molecules. As used herein, substantially homologous alsorefers to sequences showing complete identity to the specified DNA orpolypeptide sequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying the result by 100. Readily available computerprograms can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5Suppl. 3:353-358, National biomedical Research Foundation, Washington,D.C., which adapts the local homology algorithm of Smith and Waterman(1981) Advances in Appl. Math. 2:482-489 for peptide analysis. Programsfor determining nucleotide sequence identity are available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAPprograms, which also rely on the Smith and Waterman algorithm. Theseprograms are readily utilized with the default parameters recommended bythe manufacturer and described in the Wisconsin Sequence AnalysisPackage referred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

Another method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GeniBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

The term “functionally equivalent” intends that the amino acid sequenceof the CAMP factor is one that will elicit a substantially equivalent orenhanced immunological response, as defined above, as compared to theresponse elicited by a CAMP factor having identity with either themature sequence for the reference CAMP factor, or an immunogenic portionthereof.

A “heterologous” region of a DNA construct is an identifiable segment ofDNA within or attached to another DNA molecule that is not found inassociation with the other molecule in nature. Thus, when theheterologous region encodes a bacterial gene, the gene will usually beflanked by DNA that does not flank the bacterial gene in the genome ofthe source bacteria. Another example of the heterologous coding sequenceis a construct where the coding sequence itself is not found in nature(e.g., synthetic sequences having codons different from the nativegene). Allelic variation or naturally occurring mutational events do notgive rise to a heterologous region of DNA, as used herein.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from a subject, including but not limited to, forexample, blood, plasma, serum, fecal matter, urine, bone marrow, bile,spinal fluid, lymph fluid, samples of the skin, external secretions ofthe skin, respiratory, intestinal, and genitourinary tracts, tears,saliva, milk, blood cells, organs, biopsies and also samples of in vitrocell culture constituents including but not limited to conditioned mediaresulting from the growth of cells and tissues in culture medium, e.g.,recombinant cells, and cell components.

As used herein, the terms “label” and “detectable label” refer to amolecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes,metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.The term “fluorescer” refers to a substance or a portion thereof whichis capable of exhibiting fluorescence in the detectable range.Particular examples of labels which may be used under the inventioninclude fluorescein, rhodamine, dansyl, umbelliferone, Texas red,luminol, NADPH and α-β-galactosidase.

The term “treatment” as used herein refers to either (i) the preventionof infection or reinfection (prophylaxis), or (ii) the reduction orelimination of symptoms of the disease of interest (therapy).

By “vertebrate subject” is meant any member of the subphylum cordata,including, without limitation, mammals such as cattle, sheep, pigs,goats, horses, and humans; domestic animals such as dogs and cats; andbirds, including domestic, wild and game birds such as cocks and hensincluding chickens, turkeys and other gallinaceous birds; and fish. Theterm does not denote a particular age. Thus, both adult and newbornanimals, as well as fetuses, are intended to be covered.

B. General Methods

Central to the present invention is the discovery that the CAMP factoris capable of eliciting a protective immune response in a vertebratesubject. The gene for the S. uberis CAMP factor has been isolated andcharacterized and the CAMP factor encoded thereby sequenced. The proteinproduct from the S. uberis CAMP factor gene has been shown to protectcattle from subsequent challenge with S. uberis. Additionally, chimericproteins, including CAMP factor epitopes from multiple bacterialspecies, confer protection against bacterial challenge and provide avaccine antigen which is cross-reactive with various bacterial speciesand which is more immunogenic than the individual CAMP factors.Moreover, antibodies raised against purified S. uberis CAMP factorcross-react with S. agalactiae protein B. As shown in the examples, theS. uberis CAMP factor is secreted when produced recombinantly in E.coli.

The complete coding sequence of S. uberis CAMP factor is shown in FIG. 2(SEQ ID NO:1). The S. uberis CAMP factor gene encodes a preprotein ofabout 258 amino acids (amino acid residues 1 through 258, inclusive, ofFIG. 2, SEQ ID NO:2) that includes an N-terminal signal sequenceapproximately 30 amino acids in length (see, FIG. 6B; SEQ ID NO:7). Theprecursor molecule has a calculated molecular weight of approximately28.5 kDa. The mature S. uberis CAMP factor thus includes amino acidresidues 31 through 258, inclusive, as depicted in FIG. 2. Additionally,the coding sequence of S. agalactiae CAMP factor is shown in FIG. 3 (SEQID NO:3). The S. agalactiae CAMP factor gene encodes a preprotein ofabout 255 amino acids (amino acid residues 1 through 255, inclusive, ofFIG. 3, SEQ ID NO:4) that includes an N-terminal signal sequenceapproximately 29 amino acids in length (see, FIG. 6A, SEQ ID NO:6). Asdiscussed further below, the portion of the CAMP factor gene encodingthe signal sequence can be included in constructs that encode the CAMPfactor and chimeras thereof to direct secretion of the CAMP factor uponexpression. Alternatively, such constructs can include a heterologoussignal sequence. Additionally, the CAMP factor signal sequence and thenucleic acid sequence encoding the same can be used with heterologousproteins and nucleic acid molecules, to aid in the secretion thereof.

As shown in FIG. 4, alignment of the amino acid sequence of the S.agalactiae CAMP factor with the deduced sequence of the S. uberis CAMPfactor shows an overall similarity of about 69%. Approximately 63% ofthe amino acid residues are identical. The two proteins can be separatedinto low and high homology regions. The majority of conserved aminoacids are located after the leucine residue corresponding to amino acid87 of S. agalactiae CAMP and amino acid 90 of S. uberis CAMP. The lowhomology region (also termed the N-terminal variable region herein) thusincludes residues 1-90 of S. uberis and 1-87 of S. agalactiae anddisplays an overall homology of about 49% between the two species. Insum, the first third of the two CAMP proteins includes non-identicalsignal sequences of 29 amino acids (S. agalactiae) and 30 amino acids(S. uberis), respectively, followed by regions of 57 amino acids and 59amino acids, respectively, which do not share extensive homology. Thehigh homology region found from residue 91 on of S. uberis and 88 on ofS. agalactiae, displays an overall homology of about 79%.

The exact localization and sequence of the CAMP factor gene allows forin vitro mutagenesis studies to assess the functions of differentdomains on the CAMP protein. Also, the present data permits thegeneration of stable CAMP factor-synthesis deficient mutants throughgene replacement and other molecular techniques. Comparison of thevirulence of native and mutant S. uberis strains in animals providesimportant information regarding the contribution of the CAMP factor tothe pathogenicity of bacteria expressing CAMP factors.

Epitopes derived from the various CAMP factors from multiple bacterialspecies can be used in combination in order to provide a multivalentvaccine that confers broad protection against bacterial infection, suchas mastitis. Such epitopes can be provided individually in one or moresubunit vaccine compositions, or can be conveniently provided as achimeric protein, expressed recombinantly as a fusion protein orexpressed individually and subsequently fused.

In particular, as explained above, the CAMP factor is found in a varietyof bacterial species, including, without limitation S. uberis, group Bstreptococci (GBS) such as S. agalactiae (Jürgens et al. (1985) J.Chromatogr. 348:363-370; Rühlmann et al. (1988) FEBS Lett 235:262-266;Schneewind et al. (1988) Infect. Immun. 56:2174-2179), A.pleuropneumoniae (Frey et al. (1989) Infect. Immun. 57:2050-2056), groupA streptococci (GAS), such as S. pyogenes (Gase et al. (1999) Infect.Immun. 67:4725-4731), and streptococci from serogroups C, M, P, R, andU, Listeria monocytogenes and Listeria seeligeri (Rocourt, J. andGrimont, P. A. D. (1983) Int. J. Syst. Bacteriol. 33:866-869), Aeromonassp. (Figura, N. and Guglielmetti, P. (1987) J. Clin. Microbiol.25:1341-1342), Rhodococcus equi (Fraser, G. (1961) Nature 189:246), andcertain Vibrio spp. (Kohler, W. (1988) Zentralbl. Bakteriol. Mikrobiol.Hyg. Ser. A 270:35-40). With the exception of the N-terminal region, theamino acid sequences of the CAMP factor proteins produced by the variousbacterial strains are highly conserved. Other localized variable regionsoccur throughout the CAMP factor molecule. See, for example FIG. 4.Therefore, it is desirable to construct multiple epitope CAMP factorfusion proteins comprising epitopes derived from both the highlyconserved regions of the CAMP factor, and the variable regions of theCAMP factor proteins from more than one of the above bacterial species.Experiments performed in support of the present invention havedemonstrated that such a protein is capable of eliciting immunityagainst streptococcal infection while providing the additional economicadvantage of minimizing the number of antigens present in the finalformulation, and concomitantly reducing the cost of producing theformulation.

Thus, the CAMP factor chimeras of the present invention can include, forexample, multiple epitopes derived from the N-terminal variable regionof the CAMP factor, such as contiguous amino acid sequences comprisingat least about 5-10 up to about 50-90 amino acids, or any integertherebetween, from the N-terminal variable region of the CAMP factorrepresented by amino acids 1-90 of the S. uberis CAMP factor, and aminoacids 1-87 of the S. agalactiae CAMP factor, from any of the variousbacterial species described herein, preferably 20 or more contiguousamino acids from this region, more preferably about 30-80, and even morepreferably 40-60 contiguous amino acids from this region. Preferably,the chimeras will include epitopes from the N-terminal variable regionfrom more than one streptococcal CAMP factor, such as from S. uberis andS. agalactiae. The chimeras optionally include additional epitopes fromother regions of the CAMP factor and may include the remainder of theCAMP factor molecule from at least one bacterial species, and optionallymore than one bacterial species. Additionally, multiple epitopes fromthe same species can be present.

Epitopes for use in the CAMP factor chimeras of the present inventioncan be readily identified by aligning the sequences of CAMP factorsfrom, e.g., two or more of the bacterial species listed above, andsearching for the variable and conserved regions. Normally, it isdesirable to include epitopes from the variable regions of the CAMPfactors in order to confer broad-based protection against a variety ofbacteria. Additional epitopes can be identified using techniques wellknown in the art, such as using standard antigenicity and hydropathyplots, for example those calculated using, e.g., the Omiga version 1.0software program available from the Oxford Molecular Group. Thiscomputer program employs the Hopp/Woods method, Hopp et al., Proc. Natl.Acad. Sci USA (1981) 78:3824-3828 for determining antigenicity profiles,and the Kyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982)157:105-132 for hydropathy plots. This program can be used with thefollowing parameters: averaging results over a window of 7; determiningsurface probability according to Emini; chain flexibility according toKarplus-Schulz; antigenicity index according to Jameson-Wolf; secondarystructure according to Garnier-Osguthorpe-Robson; secondary structureaccording to Chou-Fasman; and identifying predicted glycosylation sites.One of skill in the art can readily use the information obtained incombination with teachings of the present specification to identifyantigenic regions which may be employed in constructing the chimericproteins of the invention.

Generally, the various epitopes will be separated by spacer sequences.The spacer sequence is typically an amino acid sequence of from 1-500amino acids, preferably 1-100 amino acids, more preferably 1-50 aminoacids, preferably 1-25 amino acids, and most preferably 1-10 aminoacids, or any integer between 1-500. The spacer amino acids may be thesame or different between the various epitopes. Particularly preferredamino acids for use as spacers are amino acids with small side groups,such as alanine, glycine and valine.

The chimeras of the present invention may also include a signalsequence. The signal sequence can be a CAMP factor signal sequence, suchas either of the signal sequences depicted in FIGS. 6A and 6B, or can beany of various heterologous sequences. Non-limiting examples ofparticularly suitable signal sequences include the E. coli LipoF signalsequence, and the OmpF signal sequence. The LipoF signal sequence aidsin efficient secretion from the bacterial host cell and becomes bound tothe host cell membrane via the lipid-moiety. The protein may then beextracted from the cell surface via differential solubilization with adetergent such as Sarkosyl or TritonX-100 (see the examples). Additionalsignal sequences for use herein are discussed further below.

An especially preferred embodiment of the present invention is a chimerawhich includes one or more epitopes from any of the various CAMP factorsdescribed herein, interposed within a full-length CAMP factor backbonefrom a different bacterium than a bacterium from which at least one ofthe epitopes is obtained, if multiple epitopes are used. The backbonemay or may not include the signal sequence from the CAMP factor inquestion. Preferably, the epitope is from the N-terminal variable regionof the CAMP factor represented by residues 1-90 of S. uberis and 1-87 ofS. agalactiae, respectively. It is to be understood that this epitopecan be derived from other streptoccoal and bacterial species thatproduce the CAMP factor with corresponding N-terminal variable regions.Moreover, by “interposed” is meant that the epitope is insertedsomewhere internal to the full-length sequence and the term does notexclude the presence of spacer sequences between the epitope and the—andC-terminal regions of the full-length CAMP factor between which theepitope is inserted.

Such a construct is represented by the chimera shown at positions 27-314of FIG. 8, termed “CAMP-3” herein. Amino acids 1-26 of FIG. 8 representthe E. coli LipoF signal sequence. Amino acids 27-86 in FIG. 8correspond to amino acids 31-90 of the S. uberis CAMP factor sequenceshown in FIG. 2 (amino acids 1-60, numbered relative to the mature S.uberis sequence). Amino acid 87 is a linker amino acid. Amino acids88-144 of FIG. 8 correspond to amino acids 31-87 of the S. agalactiaesequence shown in FIG. 3 (amino acids 2-58, numbered relative to themature S. agalactiae sequence. Amino acids 145 and 146 of FIG. 8 arespacer amino acids. Amino acids 147-314 shown in FIG. 8 correspond toamino acids 91-258 of the S. uberis CAMP factor sequence shown in FIG. 2(amino acids 63-230, numbered relative to the mature S. uberissequence). Thus, the chimera shown in FIG. 8 includes a full-length S.uberis backbone with an epitope from the N-terminal sequence of the CAMPfactor from S. agalactiae inserted therein.

The CAMP factors, immunogenic fragments thereof or chimeric proteinsincluding the same, as described above, can be provided in subunitvaccine compositions. In addition to use in vaccine compositions, theproteins or antibodies thereto can be used as diagnostic reagents todetect the presence of infection in a vertebrate subject. Similarly, thegenes encoding the proteins can be cloned and used to design probes todetect and isolate homologous genes in other bacterial strains.

The vaccine compositions of the present invention can be used to treator prevent a wide variety of bacterial infections in vertebratesubjects. For example, vaccine compositions including CAMP factors fromS. uberis, group B streptococci (GBS), such as S. agalactiae and/orgroup A streptococci (GAS), e.g., S. pyogenes, and streptococci fromserogroups C, M, P, R, and U, can be used to treat streptococcalinfections in vertebrate subjects that are caused by these species. Forexample, S. uberis and S. agalactiae are common bacterial pathogensassociated with mastitis in bovine, equine, ovine and goat species.Additionally, group B streptococci, such as S. agalactiae, are known tocause numerous other infections in vertebrates, including septicemia,meningitis, bacteremia, impetigo, arthritis, urinary tract infections,abscesses, spontaneous abortion etc. Hence, vaccine compositionscontaining streptococcal CAMP factors will find use in treating and/orpreventing a wide variety of streptococcal infections.

Similarly, CAMP factors derived from Listeria monocytogenes and L.seeligeri, Aeromonas sp., Rhodococcus equi, and Vibrio spp. will finduse for treating bacterial infections caused by these species. Forexample, CAMP factors can be used to prevent or treat listeriosis in awide range of animals and birds, including humans. The infection canmanifest itself as encephalitis and meningoencephalitis in ruminants andavian species such as geese, chickens, turkeys, ducks, canaries andparrots; septicemia in monogastric animals, neonatal ruminants andpoultry; and spontaneous abortion and latent infections in a widevariety of animals. Aeromonas causes infections in fish, including insalmonids, aquarium fish, goldfish, freshwater and marine fish; as wellas infections in caged birds and in amphibians and reptiles. Rhodococcusequi causes respiratory infections, lymphangitis, peritonitis,enteritis, abscesses and spontaneous abortion in horses. Vibrio causesvibriosis in many cultured, aquarium and wild marine and estuarine fish;infections of open wounds in cetaceans; and avian vibrionic hepatitisand avian infectious hepatitis in chickens.

Thus, it is readily apparent that CAMP factor vaccines can be used totreat and/or prevent a wide variety of bacterial infections in numerousspecies.

CAMP factors from various species can be used either alone or incombination in the vaccine compositions of the present invention. Forexample, as explained above, it will sometimes be preferable to havemore than one epitope of one or more of the CAMP factors in the vaccinecompositions of the present invention so that the subject in questioncan be provided with a broad spectrum of protection against infection.In its simplest form, this can be achieved by employing a polypeptideencoding the complete sequence of one of the CAMP factors, or byemploying a combination of polypeptides encoding the sequences of two ormore of the CAMP factors or epitopes of the CAMP factors. Thus, thevaccine compositions could comprise, for example various combinations ofone or more of the CAMP factors, or a combination of several of the CAMPfactors, or even several epitopes derived from the CAMP factors, such asin CAMP factor chimeras described above.

Furthermore, the vaccine compositions of the present invention caninclude other bacterial, fungal, viral or protozoal antigens. Theseantigens can be provided separately of even as fusion proteinscomprising fragments of one or more of the CAMP factors fused to theseantigens.

Production of the CAMP Factors

The above described CAMP factors and active fragments, analogs andchimeric proteins derived from the same, can be produced by a variety ofmethods. Specifically, the CAMP factors can be isolated directly frombacteria which express the same. This is generally accomplished by firstpreparing a crude extract which lacks cellular components and severalextraneous proteins. The desired proteins can then be further purifiedi.e. by column chromatography, HPLC, immunoadsorbent techniques or otherconventional methods well known in the art.

More particularly, techniques for isolating CAMP factors have beendescribed in e.g., Skalka, B. and Smola, J. (1981) Zbl. Bakt. Hyg., I.Abt. Orig. A249:190-194; Skalka et al. (1980) Zbl. Vet. Med.B27:559-566; Skalka et al. (1979) Zbl. Vet. Med. B26:679-687; Bernheimeret al. (1979) Infect. Immun. 23:838-844; Jürgens et al. (1985) J. Chrom.348:363-370; Jürgens et al. (1987) J. Exp. Med. 165:720-732.

Alternatively, the proteins can be recombinantly produced as describedherein. As explained above, these recombinant products can take the formof partial protein sequences, full-length sequences, precursor formsthat include signal sequences, mature forms without signals, or evenfusion proteins (e.g., with an appropriate leader for the recombinanthost, or with another subunit antigen sequence for Streptococcus oranother pathogen).

The CAMP factor genes of the present invention can be isolated based onthe ability of the protein products to display CAMP activity, using CAMPassays as described below. Thus, gene libraries can be constructed andthe resulting clones used to transform an appropriate host cell.Colonies can be pooled and screened for clones having CAMP activity.Colonies can also be screened using polyclonal serum or monoclonalantibodies to the desired antigen.

Alternatively, once the amino acid sequences are determined,oligonucleotide probes which contain the codons for a portion of thedetermined amino acid sequences can be prepared and used to screen DNAlibraries for genes encoding the subject proteins. The basic strategiesfor preparing oligonucleotide probes and DNA libraries, as well as theirscreening by nucleic acid hybridization, are well known to those ofordinary skill in the art. See, e.g., DNA Cloning: Vol. I, supra;Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis, supra;Sambrook et al., supra. Once a clone from the screened library has beenidentified by positive hybridization, it can be confirmed by restrictionenzyme analysis and DNA sequencing that the particular library insertcontains the desired CAMP factor gene or a homolog thereof.

Alternatively, DNA sequences encoding the proteins of interest can beprepared synthetically rather than cloned. The DNA sequences can bedesigned with the appropriate codons for the particular amino acidsequence. In general, one will select preferred codons for the intendedhost if the sequence will be used for expression. The complete sequenceis assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence. See, e.g., Edge(1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay etal. (1984) J. Biol. Chem. 259:6311.

Once coding sequences for the desired proteins have been prepared orisolated, they can be cloned into any suitable vector or replicon.Numerous cloning vectors are known to those of skill in the art, and theselection of an appropriate cloning vector is a matter of choice.Examples of recombinant DNA vectors for cloning and host cells whichthey can transform include the bacteriophage λ (E. coli), pBR322 (E.coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coil and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus(mammalian cells). See, generally, DNA Cloning: Vols. I & II, supra;Sambrook et al., supra; B. Perbal, supra.

The gene can be placed under the control of a promoter, ribosome bindingsite (for bacterial expression) and, optionally, an operator(collectively referred to herein as “control” elements), so that the DNAsequence encoding the desired protein is transcribed into RNA in thehost cell transformed by a vector containing this expressionconstruction. The coding sequence may or may not contain a signalpeptide or leader sequence. If signal sequences are included, they caneither be the native, homologous sequences, or heterologous sequences.For example, the signal sequences for the S. uberis or S. agalactiaeCAMP factors (shown in FIGS. 6A and 6B, respectively), can be used forsecretion of various CAMP factors, as can a number of other signalsequences, well known in the art. Leader sequences can be removed by thehost in post-translational processing. See, e.g., U.S. Pat. Nos.4,431,739; 4,425,437; 4,338,397.

Other regulatory sequences may also be desirable which allow forregulation of expression of the protein sequences relative to the growthof the host cell. Regulatory sequences are known to those of skill inthe art, and examples include those which cause the expression of a geneto be turned on or off in response to a chemical or physical stimulus,including the presence of a regulatory compound. Other types ofregulatory elements may also be present in the vector, for example,enhancer sequences.

The control sequences and other regulatory sequences may be ligated tothe coding sequence prior to insertion into a vector, such as thecloning vectors described above. Alternatively, the coding sequence canbe cloned directly into an expression vector which already contains thecontrol sequences and an appropriate restriction site.

In some cases it may be necessary to modify the coding sequence so thatit may be attached to the control sequences with the appropriateorientation; i.e., to maintain the proper reading frame. It may also bedesirable to produce mutants or analogs of the CAMP factor of interest.Mutants or analogs may be prepared by the deletion of a portion of thesequence encoding the protein, by insertion of a sequence, and/or bysubstitution of one or more nucleotides within the sequence. Techniquesfor modifying nucleotide sequences, such as site-directed mutagenesis,are described in, e.g., Sambrook et al., supra; DNA Cloning, Vols. I andII, supra; Nucleic Acid Hybridization, supra.

The expression vector is then used to transform an appropriate hostcell. A number of mammalian cell lines are known in the art and includeimmortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),Madin-Darby bovine kidney (“MDBK”) cells, as well as others. Similarly,bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcusspp., will find use with the present expression constructs. Yeast hostsuseful in the present invention include inter alia, Saccharomycescerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha,Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii,Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica.Insect cells for use with baculovirus expression vectors include, interalia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophilamelanogaster, Spodoptera frugiperda, and Trichoplusia ni.

Depending on the expression system and host selected, the proteins ofthe present invention are produced by culturing host cells transformedby an expression vector described above under conditions whereby theprotein of interest is expressed. The protein is then isolated from thehost cells and purified. If the expression system secretes the proteininto the growth media, the protein can be purified directly from themedia. If the protein is not secreted, it is isolated from cell lysates.The selection of the appropriate growth conditions and recovery methodsare within the skill of the art.

The proteins of the present invention may also be produced by chemicalsynthesis such as solid phase peptide synthesis, using known amino acidsequences or amino acid sequences derived from the DNA sequence of thegenes of interest. Such methods are known to those skilled in the art.Chemical synthesis of peptides may be preferable if a small fragment ofthe antigen in question is capable of raising an immunological responsein the subject of interest.

The CAMP factors, fragments, analogs and chimeras containing the same,can be tested for CAMP activity using any of several standard tests. Forexample, CAMP factors are known to display lytic action on a variety oftarget cells including bovine and ovine erythrocytes. Thus, a convenientmethod for testing for CAMP factor activity utilizes standard hemolyticreactions using ovine or bovine erythrocytes. See, e.g., Christie et al.(1944) Aus. J. Exp. Biol. Med. Sci. 22:197-200; Brown et al. (1974)Infect. Immun. 9:377-383; Darling, C. L. (1975) J. Clin. Microbiol.1:171; Wilkinsin, H. W. (1977) J. Clin. Microbiol 6:42; Bernheimer etal. (1979) Infect. Immun. 23:838-844; Skalka, B. and Smola, J. (1981)Zbl. Bakt. Hyg., I. Abt. Orig. A249:190-194; Huser et al. (1983) J. Gen.Microbiol. 129:1295.

Activity can also be tested by monitoring the release of entrappedmarker molecules from liposomes made from materials susceptible todisruption by CAMP factors. For example, CAMP activity can be monitoredusing [¹⁴C]glucose-containing liposomes prepared from, e.g.,sphingomyelin, cholesterol and dicetyl phosphate, and measuring therelease of trapped [¹⁴C]glucose due to disruption of the liposomes bythe CAMP factor. See, e.g., Bernheimer et al. (1979) Infect. Immun.23:838-844. Similarly, ATP release from liposomes in the presence ofCAMP factor can be monitored as described in Sterzik et al. (1984)“Interaction of the CAMP factor from S. agalactiae with artificialmembranes” In: Alouf et al., eds. Bacterial protein toxins, London:Academic Press Inc., 1984:195-196; and Sterzik et al. (1985) Zentralbl.Bakteriol. Mikrobiol. Hyg. Abt. 1 Suppl. 15:101-108. See, alsoFehrenbach et al. (1984) “Interaction of amphiphilic bacterialpolypeptides with artificial membranes.” In: Alouf et al., eds.Bacterial protein toxins, London: Academic Press Inc., 1984:317-324.

The CAMP factors of the present invention or their fragments can be usedto produce antibodies, both polyclonal and monoclonal. If polyclonalantibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat,horse, etc.) is immunized with an antigen of the present invention, orits fragment, or a mutated antigen. Serum from the immunized animal iscollected and treated according to known procedures. See, e.g., Jurgenset al. (1985) J. Chrom. 348:363-370. If serum containing polyclonalantibodies is used, the polyclonal antibodies can be purified byimmunoaffinity chromatography, using known procedures.

Monoclonal antibodies to the CAMP factors and to the fragments thereof,can also be readily produced by one skilled in the art. The generalmethodology for making monoclonal antibodies by using hybridomatechnology is well known. Immortal antibody-producing cell lines can becreated by cell fusion, and also by other techniques such as directtransformation of B lymphocytes with oncogenic DNA, or transfection withEpstein-Barr virus. See, e.g., M. Schreier et al., Hybridoma Techniques(1980); Hammerling et al., Monoclonal Antibodies and T-cell Hybridomas(1981); Kennett et al., Monoclonal Antibodies (1980); see also U.S. Pat.Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917;4,472,500, 4,491,632; and 4,493,890. Panels of monoclonal antibodiesproduced against the CAMP factor of interest, or fragment thereof, canbe screened for various properties; i.e., for isotype, epitope,affinity, etc. Monoclonal antibodies are useful in purification, usingimmunoaffinity techniques, of the individual antigens which they aredirected against. Both polyclonal and monoclonal antibodies can also beused for passive immunization or can be combined with subunit vaccinepreparations to enhance the immune response.

Vaccine Formulations and Administration

The CAMP factors of the present invention can be formulated into vaccinecompositions, either alone or in combination with other antigens, foruse in immunizing subjects as described below. Methods of preparing suchformulations are described in, e.g., Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., 18 Edition, 1990.Typically, the vaccines of the present invention are prepared asinjectables, either as liquid solutions or suspensions. Solid formssuitable for solution in or suspension in liquid vehicles prior toinjection may also be prepared. The preparation may also be emulsifiedor the active ingredient encapsulated in liposome vehicles. The activeimmunogenic ingredient is generally mixed with a compatiblepharmaceutical vehicle, such as, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents and pH bufferingagents.

Adjuvants which enhance the effectiveness of the vaccine may also beadded to the formulation. Adjuvants may include for example, muramyldipeptides, avridine, aluminum hydroxide, dimethyldioctadecyl ammoniumbromide (DDA), oils, oil-in-water emulsions, saponins, cytokines, andother substances known in the art.

The CAMP factor may be linked to a carrier in order to increase theimmunogenicity thereof. Suitable carriers include large, slowlymetabolized macromolecules such as proteins, including serum albumins,keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin,ovalbumin, and other proteins well known to those skilled in the art;polysaccharides, such as sepharose, agarose, cellulose, cellulose beadsand the like; polymeric amino acids such as polyglutamic acid,polylysine, and the like; amino acid copolymers; and inactive virusparticles.

The CAMP factors may be used in their native form or their functionalgroup content may be modified by, for example, succinylation of lysineresidues or reaction with Cys-thiolactone. A sulfhydryl group may alsobe incorporated into the carrier (or antigen) by, for example, reactionof amino functions with 2-iminothiolane or the N-hydroxysuccinimideester of 3-(4-dithiopyridyl propionate. Suitable carriers may also bemodified to incorporate spacer arms (such as hexamethylene diamine orother bifunctional molecules of similar size) for attachment ofpeptides.

Other suitable carriers for the CAMP factors of the present inventioninclude VP6 polypeptides of rotaviruses, or functional fragmentsthereof, as disclosed in U.S. Pat. No. 5,071,651, incorporated herein byreference. Also useful is a fusion product of a viral protein and thesubject immunogens made by methods disclosed in U.S. Pat. No. 4,722,840.Still other suitable carriers include cells, such as lymphocytes, sincepresentation in this form mimics the natural mode of presentation in thesubject, which gives rise to the immunized state. Alternatively, theproteins of the present invention may be coupled to erythrocytes,preferably the subject's own erythrocytes. Methods of coupling peptidesto proteins or cells are known to those of skill in the art.

Furthermore, the CAMP factors (or complexes thereof) may be formulatedinto vaccine compositions in either neutral or salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the active polypeptides) and whichare formed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed from free carboxyl groups may alsobe derived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine, and the like.

Injectable vaccine formulations will contain a “therapeuticallyeffective amount” of the active ingredient, that is, an amount capableof eliciting an immune response in a subject to which the composition isadministered. In the treatment and prevention of mastitis, for example,a “therapeutically effective amount” would preferably be an amount whichcontrols infection, as measured by, e.g. the ability of the compositionto retain or bring the somatic cell count in milk below about 500,000cells per ml. The exact amount is readily determined by one skilled inthe art using standard tests. The CAMP factor will typically range fromabout 1% to about 95% (w/w) of the composition, or even higher or lowerif appropriate. With the present vaccine formulations, 20 to 500 μg ofactive ingredient per ml of injected solution should be adequate toraise an immunological response when a dose of 1 to 3 ml per animal isadministered.

To immunize a subject, the vaccine is generally administeredparenterally, usually by intramuscular injection. Other modes ofadministration, however, such as subcutaneous, intraperitoneal andintravenous injection, are also acceptable. The quantity to beadministered depends on the animal to be treated, the capacity of theanimal's immune system to synthesize antibodies, and the degree ofprotection desired. Effective dosages can be readily established by oneof ordinary skill in the art through routine trials establishing doseresponse curves. The subject is immunized by administration of thevaccine in at least one dose, and preferably two doses. Moreover, theanimal may be administered as many doses as is required to maintain astate of immunity to infection.

Additional vaccine formulations which are suitable for other modes ofadministration include suppositories and, in some cases, aerosol,intranasal, oral formulations, and sustained release formulations. Forsuppositories, the vehicle composition will include traditional bindersand carriers, such as, polyalkaline glycols, or triglycerides. Suchsuppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10% (w/w), preferablyabout 1% to about 2%. Oral vehicles include such normally employedexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium, stearate, sodium saccharin cellulose, magnesiumcarbonate, and the like. These oral vaccine compositions may be taken inthe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations, or powders, and contain from about 10% to about95% of the active ingredient, preferably about 25% to about 70%.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

Controlled or sustained release formulations are made by incorporatingthe protein into carriers or vehicles such as liposomes, nonresorbableimpermeable polymers such as ethylenevinyl acetate copolymers andHytrel® copolymers, swellable polymers such as hydrogels, or resorbablepolymers such as collagen and certain polyacids or polyesters such asthose used to make resorbable sutures. The CAMP factors can also bedelivered using implanted mini-pumps, well known in the art.

The CAMP factors of the instant invention can also be administered via acarrier virus which expresses the same. Carrier viruses which will finduse with the instant invention include but are not limited to thevaccinia and other pox viruses, adenovirus, and herpes virus. By way ofexample, vaccinia virus recombinants expressing the novel proteins canbe constructed as follows. The DNA encoding the particular protein isfirst inserted into an appropriate vector so that it is adjacent to avaccinia promoter and flanking vaccinia DNA sequences, such as thesequence encoding thymidine kinase (TK). This vector is then used totransfect cells which are simultaneously infected with vaccinia.Homologous recombination serves to insert the vaccinia promoter plus thegene encoding the instant protein into the viral genome. The resultingTK recombinant can be selected by culturing the cells in the presence of5-bromodeoxyuridine and picking viral plaques resistant thereto.

An alternative route of administration involves gene therapy or nucleicacid immunization. Thus, nucleotide sequences (and accompanyingregulatory elements) encoding the subject CAMP factors can beadministered directly to a subject for in vivo translation thereof.Alternatively, gene transfer can be accomplished by transfecting thesubject's cells or tissues ex vivo and reintroducing the transformedmaterial into the host. DNA can be directly introduced into the hostorganism, i.e., by injection (see International Publication No.WO/90/11092; and Wolff et al. (1990) Science 247:1465-1468).Liposome-mediated gene transfer can also be accomplished using knownmethods. See, e.g., Hazinski et al. (1991) Am. J. Respir. Cell Mol.Biol. 4:206-209; Brigham et al. (1989) Am. J. Med. Sci. 298:278-281;Canonico et al. (1991) Clin. Res. 39:219A; and Nabel et al. (1990)Science 1990) 249:1285-1288. Targeting agents, such as antibodiesdirected against surface antigens expressed on specific cell types, canbe covalently conjugated to the liposomal surface so that the nucleicacid can be delivered to specific tissues and cells susceptible toinfection.

Diagnostic Assays

As explained above, the CAMP factors, variants and immunogenic fragmentsthereof and chimeras comprising CAMP factor epitopes, may also be usedas diagnostics to detect the presence of reactive antibodies ofstreptococcus, for example S. uberis, S. agalactiae and/or S.dysgalactiae, in a biological sample in order to determine the presenceof streptococcus infection. For example, the presence of antibodiesreactive with a CAMP factor can be detected using standardelectrophoretic and immunodiagnostic techniques, including immunoassayssuch as competition, direct reaction, or sandwich type assays. Suchassays include, but are not limited to, Western blots; agglutinationtests; enzyme-labeled and mediated immunoassays, such as ELISAs;biotin/avidin type assays; radioimmuno assays; immunoelectrophoresis;immunoprecipitation, etc. The reactions generally include revealinglabels such as fluorescent, chemiluminescent, radioactive, enzymaticlabels or dye molecules, or other methods for detecting the formation ofa complex between the antigen and the antibody or antibodies reactedtherewith.

The aforementioned assays generally involve separation of unboundantibody in a liquid phase from a solid phase support to whichantigen-antibody complexes are bound. Solid supports which can be usedin the practice of the invention include substrates such asnitrocellulose (e.g., in membrane or microtiter well form);polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex(e.g., beads or microtiter plates); polyvinylidine fluoride; diazotizedpaper; nylon membranes; activated beads, magnetically responsive beads,and the like.

Typically, a solid support is first reacted with a solid phase component(e.g., one or more CAMP factors) under suitable binding conditions suchthat the component is sufficiently immobilized to the support.Sometimes, immobilization of the antigen to the support can be enhancedby first coupling the antigen to a protein with better bindingproperties. Suitable coupling proteins include, but are not limited to,macromolecules such as serum albumins including bovine serum albumin(BSA), keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, and other proteins well known to those skilledin the art. Other molecules that can be used to bind the antigens to thesupport include polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and the like. Suchmolecules and methods of coupling these molecules to the antigens, arewell known to those of ordinary skill in the art. See, e.g., Brinkley,M. A. Bioconjugate Chem. (1992) 3:2-13; Hashida et al., J. Appl.Biochem. (1984) 6:56-63; and Anjaneyulu and Staros, International J. ofPeptide and Protein Res. (1987) 30:117-124.

After reacting the solid support with the solid phase component, anynon-immobilized solid-phase components are removed from the support bywashing, and the support-bound component is then contacted with abiological sample suspected of containing ligand moieties (e.g.,antibodies toward the immobilized antigens) under suitable bindingconditions. After washing to remove any non-bound ligand, a secondarybinder moiety is added under suitable binding conditions, wherein thesecondary binder is capable of associating selectively with the boundligand. The presence of the secondary binder can then be detected usingtechniques well known in the art.

More particularly, an ELISA method can be used, wherein the wells of amicrotiter plate are coated with a CAMP factor protein. A biologicalsample containing or suspected of containing anti-CAMP factorimmunoglobulin molecules is then added to the coated wells. After aperiod of incubation sufficient to allow antibody binding to theimmobilized antigen, the plate(s) can be washed to remove unboundmoieties and a detectably labeled secondary binding molecule added. Thesecondary binding molecule is allowed to react with any captured sampleantibodies, the plate washed and the presence of the secondary bindingmolecule detected using methods well known in the art.

Thus, in one particular embodiment, the presence of bound anti-CAMPfactor ligands from a biological sample can be readily detected using asecondary binder comprising an antibody directed against the antibodyligands. A number of anti-bovine immunoglobulin (Ig) molecules are knownin the art which can be readily conjugated to a detectable enzyme label,such as horseradish peroxidase, alkaline phosphatase or urease, usingmethods known to those of skill in the art. An appropriate enzymesubstrate is then used to generate a detectable signal. In other relatedembodiments, competitive-type ELISA techniques can be practiced usingmethods known to those skilled in the art.

Assays can also be conducted in solution, such that the CAMP factorproteins and antibodies specific for those proteins form complexes underprecipitating conditions. In one particular embodiment, CAMP factorproteins can be attached to a solid phase particle (e.g., an agarosebead or the like) using coupling techniques known in the art, such as bydirect chemical or indirect coupling. The antigen-coated particle isthen contacted under suitable binding conditions with a biologicalsample suspected of containing antibodies for the CAMP factor proteins.Cross-linking between bound antibodies causes the formation ofparticle-antigen-antibody complex aggregates which can be precipitatedand separated from the sample using washing and/or centrifugation. Thereaction mixture can be analyzed to determine the presence or absence ofantibody-antigen complexes using any of a number of standard methods,such as those immunodiagnostic methods described above.

In yet a further embodiment, an immunoaffinity matrix can be provided,wherein a polyclonal population of antibodies from a biological samplesuspected of containing anti-CAMP factor molecules is immobilized to asubstrate. In this regard, an initial affinity purification of thesample can be carried out using immobilized antigens. The resultantsample preparation will thus only contain anti-streptococcus moieties,avoiding potential nonspecific binding properties in the affinitysupport. A number of methods of immobilizing immunoglobulins (eitherintact or in specific fragments) at high yield and good retention ofantigen binding activity are known in the art. Not being limited by anyparticular method, immobilized protein A or protein G can be used toimmobilize immunoglobulins.

Accordingly, once the immunoglobulin molecules have been immobilized toprovide an immunoaffinity matrix, labeled CAMP factor proteins arecontacted with the bound antibodies under suitable binding conditions.After any non-specifically bound antigen has been washed from theimmunoaffinity support, the presence of bound antigen can be determinedby assaying for label using methods known in the art.

Additionally, antibodies raised to the CAMP factor proteins, rather thanthe CAMP factors themselves, can be used in the above-described assaysin order to detect the presence of antibodies to the proteins in a givensample. These assays are performed essentially as described above andare well known to those of skill in the art.

The above-described assay reagents, including the CAMP factor proteins,or antibodies thereto, can be provided in kits, with suitableinstructions and other necessary reagents, in order to conductimmunoassays as described above. The kit can also contain, depending onthe particular immunoassay used, suitable labels and other packagedreagents and materials (i.e. wash buffers and the like). Standardimmunoassays, such as those described above, can be conducted usingthese kits.

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Deposits of Strains Useful in Practicing the Invention

A deposit of biologically pure cultures of the following strains wasmade with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va., under the provisions of the Budapest Treaty.The accession number indicated was assigned after successful viabilitytesting, and the requisite fees were paid. The designated deposits willbe maintained for a period of thirty (30) years from the date ofdeposit, or for five (5) years after the last request for the deposit,whichever is longer. Should a culture become nonviable or beinadvertently destroyed, or, in the case of plasmid-containing strains,lose its plasmid, it will be replaced with a viable culture(s) of thesame taxonomic description.

Should there be a discrepancy between the sequence presented in thepresent application and the sequence of the gene of interest in thedeposited plasmid due to routine sequencing errors, the sequence in thedeposited plasmid controls.

Strain Deposit Date ATCC No. pJLD21 in E. coli JF1754 Jun. 9, 1995 69837C. Experimental

Materials and Methods

Enzymes were purchased from commercial sources, and used according tothe manufacturers' directions. Radionucleotides and nitrocellulosefilters were also purchased from commercial sources.

In the isolation of DNA fragments, except where noted, all DNAmanipulations were done according to standard procedures. See, Sambrooket al., supra. Restriction enzymes, T₄ DNA ligase, E. coli, DNApolymerase I, Klenow fragment, and other biological reagents can bepurchased from commercial suppliers and used according to themanufacturers' directions. Double stranded DNA fragments were separatedon agarose gels.

Bacterial Strains, Plasmids and Growth Conditions:

Except where indicated, the E. coli strain used for cloning the S.uberis CAMP factor gene was JF1754 (hsdR lac gal metB leuB hisB)(McNeil, J. B. and Friesen, J. D. (1981) Mol. Gen. Genet. 184:386-393).Competent E. coli JF1754 was made as previously described (Hanahan, D.“Techniques for transformation of E. coli.” In: Glover D M, ed. DNAcloning (Volume I): a practical approach. Oxford: IRL Press,1985:109-135). E. coli strains used for production of the chimeric CAMPfactor were XLI Blue MRF (recA1, endA1, gyrA96, thi-1, hsdR17, supE44,relA1, lac[F′ proAB lacI^(q)ZΔM15 Tn10(TetR] (Stratagene, La Jolla,Calif.); and J5, ATCC Accession No. 43745, J. Clin. Microbiol (1987)25:1009-1013.

E. coli cells were grown in Luria broth (Difco Laboratories) or onLuria-agar (Difco Laboratories) plates. Ampicillin was used at 50 μg/mlfor the growth of E. coli strains containing recombinant plasmids. FourS. uberis strains, as well as S. agalactiae and S. aureus, were obtainedfrom the American Type Culture Collection (ATCC Accession Nos. 9927,13386, 13387, 19436, 27541 and 25923, respectively). Other S. uberisstrains are field isolates kindly provided by M. Chirino-Trejo,University of Saskatchewan. All streptococcal strains were grown inbrain heart infusion broth (BHI, Difco Laboratories) or on base #2 bloodagar plates with 5% sheep blood (PML microbiologicals).

The cloning vector pTZ18R (Mead et al. (1986) Protein Eng. 1:67-74) wasobtained from Pharmacia Canada Ltd. The cloning vectors used forpreparing the chimeric CAMP constructs were pAA556a and pET15-b.

Preparation of S. aureus Beta-Toxin:

S. aureus was cultured in BHI for 18 h at 37° C. and the supernatantobtained after centrifugation at 5,000 g was sterilized by filtrationthrough a 0.22-μM filter (Nalge company). This material, referred to ascrude beta-toxin, was stored at −20° C.

CAMP Reaction

Bacteria were screened for CAMP activity as described (Schneewind et al.(1988) Infect. Immun. 56:2174-2179). Briefly, strains were streakedperpendicular to a streak of beta-toxin-producing S. aureus on bloodagar plates and after 6 h-20 h incubation at 37° C., they were observedfor hemolysis.

Purification of CAMP Factor

CAMP factor was partially purified from the culture supernatant of S.uberis (ATCC Accession No. 9927) by Octyl-Sepharose CL-4B (Pharmacia)chromatography as described by Jürgens et al. (1985) J. Chrom.348:363-370.

Polyclonal Antibodies

To analyze the recombinant CAMP factor of S. uberis, polyclonalantibodies directed against the purified CAMP factor were obtained. Micewere immunized by intraperitoneal injection of 20 μg of the purifiedCAMP protein with complete Freund's adjuvant. This primary immunizationwas followed 3 weeks later by the second intraperitoneal injection ofthe same amount of CAMP protein with incomplete Freund adjuvant andanother 3 weeks later by the third intravenous injection of 20 μg ofCAMP protein with incomplete Freund adjuvant. The blood serum sampleswere then taken 10 days later.

PAGE and Immunoblotting

Protein samples of S. agalactiae and E. coli were obtained from culturesupernatants by trichloroacetic acid (TCA)-precipitation at a finalconcentration of 10%. SDS-polyacrylamide gel electrophoresis (PAGE) ofproteins was performed as described by Laemmli (Laemmli, U. K. (1970)Nature 227:680-685). Proteins were electroblotted onto nitrocellulosemembranes as recommended by the supplier (Bio-Rad) and the blots weredeveloped as described elsewhere Theisen, M. and Potter, A. A. (1992) J.Bacteriol. 174:17-23) with the following differences. The firstantiserum used was mouse polyclonal antiserum against partially-purifiedS. uberis CAMP protein, and it was absorbed with antigens of the E. colihost strain as described previously (Frey et al. (1989) Infect. Immun.57:2050-2056). The second antibody used in blotting procedure was thegoat anti-mouse IgG coupled to alkaline phosphatase (Kirkegaard & PerryLaboratories, Inc.).

DNA Manipulations

All molecular techniques were as recommended by the supplier (PharmaciaCanada Ltd.) or Sambrook et al., supra. Chromosomal DNA of S. uberis wasprepared from cells grown in 100 ml BHI plus 5% (w/v) glycine. Cellswere pelleted and resuspended in 2.5 ml of TES buffer (30 mM Tris-HCl, 5mM EDTA, 50 mM NaCl; pH 8.0) with 25% sucrose and 1.6 mg/ml lysozyme(Sigma). The suspension was incubated for 1 h at 37° C., followed byfreezing at −70° C. The frozen cells were thawed in a 65° C. water bath.EDTA and proteinase K (Pharmacia) were added to final concentrations of20 mM and 1.2 mg/ml, respectively, before incubation at 65° C. for 30min. To lyse cells completely, sarkosyl was added to 1% and incubated at37° C. for 1 h. Two ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8.9)was added prior to phenol: chloroform extraction. DNA was recovered byethanol precipitation and was treated with RNase (Pharmacia CanadaLtd.).

Size-fractionated Sau3AI-digested chromosomal DNA fragments wereisolated by sucrose density gradient centrifugation (Sambrook et al.,supra).

DNA sequence was determined by the dideoxy-chain termination method ofSanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467 ondouble-stranded plasmid templates by using a^(T7) Sequencing kit(Pharmacia Canada Ltd.).

RNA Analyses

RNA from E. coli strains was isolated as described previously (Lloubeset al. (1986) Nucleic Acid Res. 14:2621-2636) with an additionalRNase-free DNase I digestion. RNA from S. uberis was prepared asfollows. The cell pellet from a 10 ml culture (OD₆₀₀=0.6) wasresuspended in 250 μl of TE buffer (pH 8.0) containing 500 u ofmutanolysin (Sigma) and incubated at 37° C. for 30 min. Lysis buffer(250 μl)(60 mM Tris-HCl pH 7.4, 200 mM NaCl, 10 mM EDTA, 2% SDS) and 100μg/ml (final concentration) of proteinase K was added and the incubationcontinued for 1 h. The sample was extracted once with 65° C. phenol(water saturated, pH 4.0) and twice with room temperature phenol. RNAwas recovered by ethanol precipitation and treated with DNase I(Pharmacia Canada Ltd.).

Primer extension assay was performed as described by Miller et al.(1986) Nucleic Acids Res. 14:7341-60. RNasin and moloney murine leukemiavirus reverse transcriptase were obtained from Pharmacia Canada Ltd.

EXAMPLE 1 Cloning and Expression of the S. uberis CAMP Factor Gene

Chromosomal DNA of S. uberis (ATCC 9927) was partially digested withSau3AI and size fractionated in a sucrose gradient; from this, 2- to5-kb DNA fragments were recovered. The ends of these fragments werepartially filled in with dGTP and dATP and ligated into pTZ18R which wascut with SalI and partially filled in with dTTP and dCTP. Followingtransformation of E. coli JF 1754 competent cells, clones expressing theCAMP factor gene were identified on blood plates with ampicillin andbeta-toxin on the surface. Six clones from a total of 10,000 werephenotypically hemolytic and each one mediated a distinct CAMP reaction.One of them, containing recombinant plasmid pJLD21, was selected forfurther study.

Plasmid pJLD21 contained a 5.2 kb insert fragment and the CAMP factorgene, cfu, was localized within a 3.2 kb BamHI fragment after theCAMP-positive subclone pJLD21-2 was generated (FIG. 1). This subclonewas further analyzed with more restriction enzymes for sequencingpurposes.

To study the expression of the recombinant CAMP factor, SDS-PAGEanalysis of supernatant proteins from Cfu⁺ E. coli JF1754(pJLD21) andhost E. coli JF1754 (pTZ18R) was performed. Compared to the vectorcontrol, no distinguishable band was observed in the lane containingsupernatant from the Cfu⁺ clone, indicating that either expression wasat a very low level or the protein was not secreted efficiently. Toidentify the CAMP factor encoded by pJLD21, the proteins separated bySDS-PAGE were transferred to a nitrocellulose membrane andimmunoblotted. The Cfu⁺ E. coli clone carrying pJLD21 expressed aprotein with molecular weight of 28,000, similar to the native CAMPfactor of S. uberis.

Another expression plasmid for the S. uberis CAMP factor, pGH-CAMP, wasconstructed as shown in FIG. 5. In particular, A 1.7 kb EcoRI-BamHIfragment of pJLD21-2 was filled in with Klenow polymerase and insertedinto pGH433 which was cut by BamHI and filled in a similar fashion.Plasmid pGH433 is an expression vector containing a tac promoter, atranslational start site with restriction enzyme sites allowing ligationin all three reading frames followed by stop codons in all readingframes. See, Theisen, M. and Potter, A. A. (1992) J. Bacteriol174:17-23.

The expression plasmids were used to transform E. coli JF1754 (describedabove). The CAMP factor was prepared from inclusion bodies as describedin, e.g., Rossi-Campos et al. (1992) Vaccine 10:512-518, for use in thevaccine trials below. Briefly, bacteria were grown to mid-log phase andisopropyl-β,D-thiogalactoside (IPTG) was added and the cultures wereincubated with vigorous agitation at 37° C. The bacteria were harvestedby centrifugation, resuspended and frozen at −70° C. The frozen cellswere thawed at room temperature and lysozyme was added. A detergent mixwas then added. The viscosity was reduced by sonication and proteinaggregates were harvested by centrifugation. The pellets were dissolvedin a minimal volume of 4 M guanidine hydrochloride. The proteins wereanalyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresisand the protein concentration was estimated by comparing the intensityof the coomassie blue-stained bands to a bovine serum albumin standard.

EXAMPLE 2 Nucleotide Sequence of S. uberis CAMP Factor Gene

To obtain the nucleotide sequence of the S. uberis CAMP factor gene,each of the EcoRI, HindIII, HincII and SacI fragments of pJLD21-2 wasindividually cloned into pTZ18R. Fragments were sequenced in bothorientations as shown in FIG. 1. The coding sequence for the S. uberisCAMP factor, as well as the deduced amino acid sequence, is shown inFIG. 2 (SEQ ID NOS:1 and 2).

EXAMPLE 3 Comparison of the S. uberis CAMP Factor with S. agalactiaeCAMP Factor

To compare the S. uberis CAMP factor with protein B of S. agalactiae, aconcentrated culture supernatant of S. agalactiae containing protein B(Jürgens et al. (1985) J. Chrom. 348:363-370) was separated by SDS-PAGEand analyzed by immunoblotting with antibodies against the purified S.uberis CAMP factor. A 25 kDa protein band from the S. agalactiaesupernatant reacted in the immunoblot. This data indicated thatmonospecific antibodies raised against the S. uberis CAMP factor couldcross-react with S. agalactiae protein B. This is not surprising sincealignment of the amino acid sequence of the S. agalactiae CAMP factorwith the deduced amino acids of the S. uberis CAMP factor showed 66.4%identical residues (FIG. 4).

EXAMPLE 4 Distribution of CAMP Factor Genes in Eight S. uberis Strains

To study the distribution of the CAMP factor gene in other S. uberisstrains, chromosomal DNA prepared from eight S. uberis strains wasdigested with the restriction endonuclease HindIII and separated on anagarose gel. Southern blot analysis with the 576 bp HindIII-EcoRIfragment of pJLD21 as a probe (FIG. 1) showed that a fragment identicalin size to the HindIII fragment (1.2 kb) in pJLD21 was present in threeS. uberis strains which were CAMP-reaction positive, while none of theCAMP reaction negative strains reacted with the probe. Thus, theCAMP-negative strains do not contain the cfu gene.

EXAMPLE 5 Immunogenicity and Protective Capability of the CAMP Factor

S. uberis CAMP factor, encoded by pGH-CAMP, was prepared from inclusionbodies as described in Example 1. The antigen was formulated in VSA3adjuvant which is a combination of Emulsigen Plus™ from MVPLaboratories, Ralston, Nebr. and Dimethyldioctadecyl ammonium bromide(DDA) from Kodak (Rochester, N.Y.). The final concentration was 25 μgper ml of CAMP factor, 30% Emulsigen Plus, 0.9% Tween-80, and 2.5 mg perml of DDA. The dose volume was 2 cc containing 50 μg of recombinantantigen.

Fifteen healthy lactating dairy cows from the Pennsylvania StateUniversity Mastitis Research Herd were used to study the ability of theS. uberis CAMP factor to protect cows from mastitis. Animals wereassigned to two groups of five cows. Treatment groups consisted of 1)experimental, given the vaccine including the S. uberis CAMP factor,administered intramuscularly at dry off and again 28 days later, and 2)placebo (vehicle) administered via intramuscular injection at dry offand again 28 days later.

All animals were challenged in one quarter with S. uberis on day four oflactation. Milk and blood samples were obtained as outlined in Table 1.

TABLE 1 Sampling Schedule TIME SAMPLE dry off, D − 0 serum, milk,immunization 14 days dry, D + 14 serum 28 days dry, D + 28 serum,immunization 52 days dry, D + 52 serum calving, C − 0 serum, milk,bacteriology 4 days lactation, CH − 0 serum, milk, bacteriology,challenge 5 days lactation, CH + 1 bacteriology 6 days lactation, CH + 2bacteriology 7 days lactation, CH + 3 serum, milk, bacteriology 14 dayslactation, CH + 10 serum, milk, bacteriology 21 days lactation, CH + 17serum, milk, bacteriology

The challenge strain of S. uberis (ATCC strain 9927) was obtained from aclinical case of bovine mastitis. The stock culture of S. uberis wasgrown in tryptic soy broth and individual aliquot were stored at −70° C.on blood beads until needed. The bacterial challenge was prepared byrolling the stock bead cultures onto esculin blood agar platescontaining 5% whole blood. After 24 hours incubation at 37° C., a singlecolony was used to inoculate 100 ml of Ultra High Temperaturepasteurized (UHT) milk and incubated for 12 hours at 37° C. The 24 hourculture was mixed well and a 100 μl aliquot was removed to inoculate asecond 100 ml of UHT milk. After a second 9 hour incubation at 37° C.,the culture was serially diluted in 10-fold increments using sterilesaline. The colony forming units (CFU) per ml of each dilution wasdetermined by absorbance on a spectrophotometer and confirmed by platepouring onto blood agar plates. The dilution containing 200 CFR of S.uberis per ml of saline was selected for each challenge.

Total Ig titers for CAMP factor were determined by an indirect ELISA.Immunlon-2 plates were coated with antigen in carbonate buffer. Prior touse, the plates were blocked with TBST (100 mM Tris Cl, pH 8.0; 150 mMNaCl; 0.05% Tween-20) and 3% BSA for 1 hour. After blocking, the plateswere washed with distilled water. Serum and milk samples were seriallydiluted in 3-fold increments using TBST containing 1% BSA. Rabbitantisera for S. uberis CAMP factor was also diluted and served as apositive control. Negative control samples contained TBST with 1% BSA.The diluted samples and controls were transferred to the coated platesand were incubated for 1 hour at room temperature. The plates werewashed thoroughly with distilled water and all wells were incubated witha horse radish peroxidase conjugate of goat anti-IgG diluted 1:2000 inTBST containing 1% BSA. Following a 1 hour incubation at roomtemperature, the plates were washed with distilled water. The amount ofantibody present in samples was visualized using ABT substrate. Thetiters of each sample were based on the absorbance reading at 405 nmwith a reference wavelength of 495 nm. A positive reading for sampleswas one in which the absorbance was two times the absorbance of theblank (negative control). Titers were determined by taking thereciprocal of the last dilution giving a positive reading. Consistencyamong assay plates was monitored by the absorbance reading of positivecontrols.

The results are shown in TABLES 2 and 3. As can be seen, antibody titerswere greater in the vaccinated animals than in the placebo group.

TABLE 2 Average Serum Titers Following Experimental Challenge with S.uberis Treatment Before Before After Group Immunization ChallengeChallenge Placebo A¹ 6.75 45.0 45.0 CAMP factor 3.00 819.00 445.50¹Serum titers of samples obtained from placebo immunized animalsscreened for CAMP factor.

TABLE 3 Average Lacteal Antibody Titers Following Experimental Challengewith S. uberis Treatment Before Before After Group ImmunizationChallenge Challenge Placebo A¹ 0.00  0.00  1.33 CAMP factor 8.00 288.0042.50 ¹Lacteal antibody titers of samples obtained from placeboimmunized animals screened for CAMP factor.

Somatic cell counts are a traditional measure of mastitis in cows.Accordingly, milk was assayed for somatic cells using standard assay.Results are shown in TABLE 4. As is readily apparent, immunized animalshad a somatic cell count within normal limits while the placebo grouphad cell counts indicating the presence of mastitis. Thus, the CAMPfactor vaccine was effective in preventing mastitis.

TABLE 4 Average Milk Somatic Cell Counts Following ExperimentalChallenge with S. uberis Before Challenge¹ After Challenge² TreatmentGroup 1000 cells/ml milk Placebo A 130.50 2825.25 CAMP factor 251.75 51.50 ¹Milk SCC obtained from quarters immediately prior tointramammary challenge with S. uberis ²Milk SCC obtained from quarters 3days following intramammary challenge with S. uberis

EXAMPLE 6 Cloning, Expression and Purification of a Chimeric CAMP FactorConstruct

A chimeric CAMP protein (CAMP-3) comprising epitopes from the S. uberisand S. agalactiae CAMP factors, was constructed in order to provide ahighly immunogenic, cross-reactive vaccine antigen. FIG. 7 shows theintermediate and final plasmids used to construct the CAMP-3 chimera.The DNA and amino acid sequences of the final construct are shown inFIG. 8. The construct includes DNA encoding amino acids 31-87 of the S.agalactiae sequence shown in FIG. 3, cloned between those encoding Leu₉₀and Lys₉₁ residues of S. uberis CAMP.

The published nucleotide sequences of the CAMP-encoding genes cfb(Podbielski et al. (1994) Med. Microbiol Immunol. 183:239-56 and cfx(Jiang et al. (1996) Microbiol. Path. 20:297-307), from S. agalactiaeand S. uberis respectively, were used to design PCR primers (shown inTable 5), allowing construction of the chimeric CAMP-encoding gene,camp-3. A PCR fragment encoding amino acid residues 30-90 of cfx wasamplified with the primers CAMP-1 and CAMP-2. The fragment was clonedinto the expression vector pAA556a (which containsOmpF-terminal-encoding sequences, directing export of OmpF fusionproteins to the E. coli cell surface) using the primer encoded BamHI andNdeI restriction sites, and the resulting construct was designatedpPolyCAMP-1. A second PCR fragment, encoding residues 31-87 of cfb, wasamplified with the primers CAMP-3 and CAMP-4 and cloned intopPoly-CAMP-1 using XhoI and NcoI sites to give pPolyCAMP-2. Finally, athird PCR fragment, encoding residues 91-258 of cfx, was amplified withthe primers CAMP-5 and CAMP-6 and digested with Eco47-3 and NcoI. Thisfragment was cloned into StuI/NcoI digested pPolyCAMP-2, and theresulting construct, pPolyCAMP-3, contained a chimeric gene, camp-3,encoding a protein of 317 aa, with a calculated M_(r) of 34,956 Da and apI of 5.5. This protein is the CAMP-3 chimera fused to the LipoF signalsequence. The camp-3 construct was sequenced in both directions usingthe primers 556-1 and 556-2. The DNA sequence of the chimeric constructwas determined using an ABI 373 DNA automatic sequencer (AppliedBiosystems). The DNA sequence and corresponding amino acid sequence ofthe CAMP-3 chimera is shown in FIG. 8 at DNA positions 79-942 (aminoacid positions 27-314). Nucleotide positions 1-78 (amino acid positions1-26) represent the LipoF signal sequence.

To provide enough pure protein for subsequent vaccine studies, anNdeI/BamHI fragment containing the camp-3 region of pPolyCAMP-3, minusthe OmpF signal sequence, was cloned into the expression vector pET15b(Novagen, Madison, Wis.) which had been digested with BamHI and NdeI.Cloning of the PCR product into this site results in the addition of anin-frame coding sequence for a hexahistidyl (6×His) tag to the CAMPcoding sequence. Subsequent expression yields a protein with an attachedhistidine tag, which permits purification of the protein undernon-denaturing conditions using metal chelate chromatography. Thus, thefinal construct, plasmid pET-CAMP3, encoded a (6×His)CAMP-3 fusionprotein. DNA sequence analysis of this construct revealed that theCAMP-3 ORF was not fused to the peptide encoding the histidine tag, butthat its expression was still controlled by IPTG. This construct wasused to transform E. coli strains, described above, using thepolyethylene glycol method (Kurien and Scofield (1995) Bio Techniques18:1023-1026) or by repeated washes of the cells in sterile-distilledwater as previously described (Frohelich and Scott (1991) Gene108:99-101).

TABLE 5 PCR and Sequencing Primers Primer Sequence Comments CAMP-15′-AAAAAAGGATCCAATCAAATAAATGTTAGTCAACCA-3′ Forward primer, annealing tont (SEQ ID NO:10) 91-112 of the S. uberis CAMP coding sequence, BamHIsite underlined. CAMP-25′-AAAAACCATGGCTACTCGAGATTTTCAACAGCTGAATTGCTG  AATTAAC-3′ Reverseprimer, annealing to nt (SEQ ID NO:11) 267-238 (opposite strand) of theS. uberis CAMP coding sequence. XhoI, and NcoI underlined. CAMP-35′-AAAAAACTCGAGCAAGTGACAACTCCACAAGTGG-3′ Forward primer, annealing to nt(SEQ ID NO:12) 91-102 of the S. agalactiae CAMP coding sequence. XhoIsite underlined. CAMP-4 5′-AAAAAACCATGGCTAAGGCCTTAATTTTTCCACGCTAGTAATAGCCTC-3′ Reverse primer, annealing to nt (SEQ ID NO:13) 261-235(opposite strand) of the S. agalactiae CAMP coding sequence. StuI, andNcoI sites underlined. CAMP-5 5′-AAAAAAGCGCTAAAACTTCACTTAGAGCTAATCCTG-3′Forward primer, annealing to nt. (SEQ ID NO:14) 271-751 (oppositestrand) of the S. uberis CAMP coding sequence. Eco47-3 site underlined.CAMP-6 5′-AAAAACCATGGTCATTACTGTAGAGCAGTATTTAATGCTTC-3′ Reverse primer,annealing to nt (SEQ ID NO:15) 777-751 (opposite strand) of the S.uberis CAMP coding sequence. NcoI site underlined. His-CAMP-15′-AAAAAACATATGTCCAATCAAATAAATGTTAGTCAACC-3′ Forward primer for cloninginto (SEQ ID NO:16) pET-15b. NdeI site underlined. His-CAMP-25′-TTTTTGGATCCTTACTGTAGAGCAGTATTTAATGC-3′ Reverse primer for cloninginto (SEQ ID NO:17) pET-15b. BamHI site underlined. 556-15′-GTGTGGAATTGTGAGCGG-3′ Forward primer for sequencing of (SEQ ID NO:18)cloned inserts in pAA556a, annealing to nt 1791-1808. 556-25′-CTCCCTGCCTCTGTC-3′ Reverse primer for sequencing of (SEQ ID NO:19)cloned inserts in pAA556a, annealing to nt 1979-1965 (opposite strand).

The CAMP-3 protein was purified by anion exchange chromatography of afiltered lysate of an IPTG-induced culture of BL21 (DE3) containingpET-CAMP3. Cells were collected by centrifugation at 6,000×g for 10 minat 4° C., washed in 0.1 M PBS (pH 7.2), and disrupted by sonication. Thevolume of the soluble fraction was adjusted to 650 ml with 20 mM Na₂HPO₄(pH 7.5), and filtered through a 0.22 μm filter (Millipore). Q-sepharosefast flow anion exchange resin was packed into an XK26/20 column to abed height of 13 cm (70 ml column volume). The column was equilibratedwith buffer A (20 mM Na₂HPO₄, pH 7.5), and the protein solution waspassed through at a rate of 7 ml/min. The column was washed with 7.4column volumes (CV) of buffer A, and protein was eluted with a gradientbuffer (0% buffer A-50% buffer B [buffer A+1 M NaCl, pH 7.5] over 12.85CV and from 50-100% buffer B in 3.6 CV, and finally 100% of buffer B for1.7 CV). The column eluate was monitored at 260 nm, and fractions wereconcentrated with BIOMAX-30 K filters. Analysis by SDS-PAGE and Westernblot determined that the CAMP-3 protein eluted in the breakthrough andbuffer A fractions. Densitometry of SDS-PA gels estimated protein purityto be>60%.

EXAMPLE 7 Immunization and Challenge of Lactating Cows with ChimericCAMP Factor

Experiments to test the effectiveness of vaccines comprising thechimeric CAMP factor, as well as the streptococcal protein, GapC, fromS. uberis and S. dysgalactiae, were conducted in lactating cows asfollows. A total of 99 lactating Holstein cows were screened for thepresence of serum IgG against S. uberis whole cells, GapC and CAMP. Fourgroups of 8 animals were selected for vaccination with a placebo,(6×His)GapC of S. uberis, (6×His)GapC of S. dysgalactiae and CAMP-3.Each vaccine dose (2 ml) included 100 μg/ml of purified (6×His)GapC,CAMP-3 or antigen-free placebo (0.85% (w/v) saline), and 30% VSA3 (VIDO,Saskatoon, Saskatchewan, Canada; van Drunen Littel-van den Hurk et al.(1993) Vaccine 11:25-35). Cows received 2 subcutaneous injections in theneck at 36 (day 0) and 15 days prior to challenge. Eight days beforechallenge, milk samples from each quarter were analysed for the presenceof bacteria, and infected animals were excluded from the trial.Subsequently, 6 cows from each group were challenged. Three hrs beforechallenge, teats were washed with clean, warm water, dried, and alcoholswabbed. Milk samples were collected for somatic cell counts (SCC) andbacteriology. The left udder quarters remained unchallenged as controls.Three ml of inoculum was administered by intramammary infusion to theright quarters of each animal, containing 3.0×10⁷ cfu/ml of anexponential-phase culture of S. uberis SU21 (clinical isolate obtainedfrom Animal Health Laboratory, Alberta, Canada) suspended in 0.85% (w/v)saline. Milk samples were collected from all quarters, daily for 7 dayspost-challenge, for determination of SSC and bacteriology. All sampleswere stored on ice, and analysed within 48 hrs of collection. Clinicalassessments of animals included measurement of rectal temperatures, andudder swelling (visual and palpated). A numerical score of 1 (normal) to3.5 (severe mastitis) was assigned to each animal and used as a means ofcomparing the severity of mastitis among vaccine groups. Milk qualitywas assessed by the presence of clots.

Serum IgG titers were determined at the time of first and secondvaccinations, at 8 days before challenge (day 28), and at 11 dayspost-challenge (day 47). Similarly, milk IgG titers were determined atday 21 and 43. Serum IgA titers were determined at day 21 and 47, andmilk IgA titers were determined at day 21 and 43. Round-bottomed,96-well microtiter plates (Nunc) were coated overnight at 4° C. withCAMP-3, and (6×His)GapC of S. uberis and S. dysgalactiae (100 ng/well in100 μl of carbonate buffer, pH 9.6), and blocked for 1 hr at 37° C. with200 μl of PBSTg. 100 μl of test sample was added/well, and plates wereincubated for 2 hrs at room temperature. After washing, alkalinephosphatase-conjugated goat anti-bovine IgG (H & L; Kirkegaard and PerryLabs. Inc., Gaithersburg, Md.) was added (100 μl/well), and plates wereincubated for 1 hr at room temperature. Plates were washed, and alkalinephosphatase activity was detected at 405 nm following incubation withρ-nitrophenyl phosphate in 1 M diethanolamine (pH 9.8) and 0.5 mM MgCl₂for 1.5 hrs at room temperature.

Determination of milk IgG and IgA was carried out after treating milkwith a commercially available rennin solution, as follows: one tablet ofRennet (CHR HANSEN) was dissolved in 40 ml of H₂O, and 0.1 ml of thissolution was added to 2 ml of milk and incubated at room temperature for4 hrs. Coagulated casein was pelleted by centrifugation at 3,000×g for20 min, and the middle layer was removed (the top layer comprised fat)and analysed as for serum samples. Both serum and milk titers weredetermined by the intersection of the least-square regression of theOD₄₀₅ versus logarithm of dilution with the OD₄₀₅ obtained from wellscontaining no serum.

Determination of SCC from milk samples was carried out at the PacificMilk Analysis Laboratory (Chilliwak, British Columbia). Samples werecollected in 14 ml polystyrene, round-bottomed tubes (Falcon) containinga preservative. SCC were fixed by mixing 0.5 ml of milk samples with 10μl of fixative liquid (0.2 mg/ml eosine, 3.3% formaldehyde solution) for18 hrs at 30° C. Samples were diluted 1/100in emulsifier electrolytesolution (12% ethanol, 0.02% Triton X-100, 0.1 M NaCl), and incubated at80° C. for 10 min. After cooling to room temperature, SCC weredetermined with a Coulter counter. Repeated measures analysis ofvariance of SCC among treatments, and over time, was performed using theSYSTAT 10 software package (SPSS Science, Chicago, USA).

Table 6 shows pre- and post-challenge titers, presented as thearithmetic means of the natural log transformed values of serum titersfrom all animals in each treatment group (standard deviations inparentheses). Prior to vaccination, only 4 animals showed any detectableserum IgG titer against (6×His)GapC. Following vaccination, all animalsvaccinated with (6×His)GapC showed a significant increase in both serumand milk anti-(6×His)GapC IgG titers, which consistently remained atleast 10-fold higher than the control animals, while anti-(6×His)GapCIgG titers in animals vaccinated with CAMP-3 were similar to those ofthe control group. Anti-(6×His)GapC IgG titers in milk were consistentlylower than the corresponding values in serum. However, immediately priorto challenge the increased serum and milk IgG titers in (6×His)GapCvaccinated animals, compared to control and CAMP-3 vaccinated animals,was apparent. Serum anti-(6×His)GapC IgA levels were detectable in allgroups prior to challenge, but rose significantly following challenge.Even the CAMP-3 vaccinated group showed an increase in serumanti-(6×His)GapC IgA titers, most likely resulting from exposure to thecell surface-associated GapC of the S. uberis challenge bacteria. InCAMP-3 vaccinated animals, a post-challenge increase in anti-(6×His)GapCmilk IgG titers was also observed, although a corresponding increase wasnot observed in serum IgG titers. In contrast to serum, milkanti-(6×His)GapC IgA was virtually undetectable in all groups, both pre-and post-challenge.

Following vaccination of cows with CAMP-3, there was a marked increasein serum and milk anti-CAMP IgG titers, compared to those of the controland (6×His)GapC vaccinated animals. Furthermore, in contrast toanti-(6×His)GapC IgG titers, anti-CAMP-3 titers increasedpost-challenge, whereas those for (6×His)GapC decreased slightly.Although the cause is unknown, this observation was consistentthroughout all vaccine groups; post-challenge serum anti-(6×His)GapC IgGtiters were found to have decreased, whereas the corresponding titers inmilk had increased (with the exception of the S. dysgalactiae(6×His)GapC vaccinated group). Pre-challenge, serum anti-CAMP-3 IgAtiters were higher than the equivalent serum anti-(6×His)GapC titersdetermined at the same time point (day 21), and at the time thepost-challenge serum samples were taken, anti-CAMP-3 IgA levels hadincreased in all groups, most significantly in those vaccinated withCAMP-3. In contrast, both pre- and post-challenge milk anti-CAMP-3 IgAtiters were virtually undetectable.

Following challenge with S. uberis SU21, at no point were bacteriarecovered from any animals, vaccinated or otherwise. This is consistentwith the results of a previous study (Finch et al. (1994) Infect. Immun.62:3599-3603), where no bacteria were isolated following challenge fromdairy cows vaccinated with heat-killed S. uberis, although bacteria wereisolated from the unvaccinated control animals. It is possible that inthe current study the inoculum administered was low enough to inducemastitis without causing persistent infection, even in unvaccinatedanimals. However, despite the absence of recoverable bacteria, animalsdid display clinical signs of disease, and SCC indicated thatinflammation had occurred. Therefore, the challenge was deemedsuccessful. Between vaccine groups, no significant differences wereobserved in rectal temperatures, and clinical scores determined thatthere were no significant differences in the severity of infection.Although no differences in milk yield were observed in any animals, thequality of milk was slightly affected in all groups, as discussed below.

The bovine udder is comprised of unconnected quarters, and bychallenging only 2 quarters with S. uberis an internal control wasprovided for each animal. FIG. 9 shows SCC over the course of the trial,for each particular vaccine group. On initial analysis, the reported SCCof the control group appeared somewhat variable; however, the overallrelationship was an increase of SCC over time, explained by a quadraticrelationship (p=0.02). After day 2, SCC of the control group increasedmarkedly, reaching their highest level at day 4 post-challenge. SCC thendecreased slightly, before rising markedly again by day 7post-challenge. This somewhat erratic trend is consistent with SCCvalues reported elsewhere, following challenge of lactating cows with S.uberis (Finch et al. (1997) Vaccine 15:1138-1143; Finch et al. (1994)Infect. Immun. 62:3599-3603). The SCC of S. dysgalactiae (6×His)GapCvaccinated animals increased sharply immediately post-challenge,reaching a maximum at day 3, before decreasing erratically over theremainder of the trial. Nevertheless, at no point was the decrease inSCC statistically significantly different from that of the controlgroup, despite an apparent difference from day 4 post-challenge onward.This result may be because the S. dysgalactiae (6×His)GapC is not asprotective as that of S. uberis.

Vaccination with S. uberis (6×His)GapC resulted in a significantdecrease in SCC, compared to the control group. From day 3 onward, SCCin this group were statistically significantly lower than those of thecontrol group (p values of 0.023 at day 3, 0.001 at day 4, 0.011 at day5, 0.006 at day 6, and 0.000 at day 7 post-challenge). SCC of cowsvaccinated with the CAMP-3 antigen were slightly higher than those ofthe S. uberis (6×His)GapC vaccinated animals, although they were stillclearly lower than those of the control group. Comparison of SCC of thecontrol and CAMP-3 vaccinated groups revealed statistically significantdifferences at days 3 (p value of 0.033), 6 and 7 post-challenge (pvalues of 0.032, and 0.046 respectively), but not at days 4 and 5, eventhough SCC were obviously lower in the CAMP-3 vaccinated group on thesedays.

Following challenge with SU21, the time that milk quality remainedaffected varied between vaccine groups. Post-challenge, milk quality inthe control, S. dysgalactiae (6×His)GapC, CAMP-3, and S. uberis(6×His)GapC vaccinated groups was reduced for a total of 21, 24, 11, and9 days respectively. According to this data, mastitis in the S.dysgalactiae (6×His)GapC vaccinated group was no less severe, if notworse, than that of the control group. Conversely, although vaccinationwith S. uberis (6×His)GapC did not completely prevent reduced milkquality, it did significantly reduce the length of time that milkquality was affected. Vaccination with CAMP-3 also appeared to reducethe length of time that milk quality was reduced, although not as muchas in the S. uberis (6×His)GapC group, which is in keeping with the SCCresults.

TABLE 6 Anti-GapC and anti-CAMP IgG and IgA titers^(a) IgG titers IgAtiters Antigen Group Serum Milk Serum Milk GapC Pre- 1 8.33 (±0.87) 4.28(±0.55) 2.75 (±0.85) 0.86 (±1.10) challenge 2 12.74 (±1.64)  7.12(±0.34) 3.39 (±0.31) 2.11 (±1.22) 3 13.21 (±0.84)  7.85 (±1.09) 2.72(±1.50) 1.24 (±1.33) 4 9.41 (±0.69) 4.51 ± 0.61 2.09 (±1.03) 1.34(±1.26) Post- 1 2.42 (±3.75) 6.38 (±0.55) 4.20 (±0.97) 1.35 (±1.55)challenge 2 10.12 (±1.34)  6.66 (±0.22) 4.78 (±0.94) 1.14 (±1.25) 310.79 (±1.07)  9.05 (±1.64) 4.64 (±1.02) 1.31 (±1.22) 4 5.30 (±4.14)5.84 (±0.46) 3.49 (±3.28) 1.80 (±1.25) CAMP Pre- 1 7.35 (±1.00) 4.06(±0.39) 1.63 (±1.53) 0.60 (±0.81) challenge 2 7.98 (±0.98) 5.33 (±0.34)0.90 (±1.36) 1.77 (±0.90) 3 7.21 (±0.99) 5.06 (±0.70) 2.56 (±1.90) 1.67(±1.42) 4 11.82 (±0.59)  8.37 (±1.02) 3.69 (±2.31) 1.46 (±1.44) Post- 16.19 (±4.82) 4.87 (±1.37) 0 0.58 (±0.94) challenge 2 6.98 (±3.52) 5.07(±0.78) 1.46 (±2.42) 0.41 (±0.99) 3 6.41 (±5.24) 5.26 (±1.00) 2.66(±2.24) 1.85 (±1.42) 4 13.47 (±0.55)  8.90 (±1.13) 5.09 (±1.42) 1.90(±1.30) ^(a)Groups shown are 1. Control, 2. S. dysgalactiae (6xHis)GapC,3. S. uberis (6xHis)GapC, and 4. CAMP-3 vaccinates. Pre-challenge datacorrespond to serum IgG titers at day 28, and serum IgA, milk IgG andIgA titers at day 21. Post-challenge data correspond to serum IgG titersat day 47, and serum IgA, milk IgG and IgA titers at day 43.

Thus, immunogenic CAMP factors are disclosed, as are methods of makingand using the same. Although preferred embodiments of the subjectinvention have been described in some detail, it is understood thatobvious variations can be made without departing from the spirit and thescope of the invention as defined by the appended claims.

1. An isolated immunogenic polypeptide comprising cohemolytic (CAMP)factor epitopes from more than one bacterial species, wherein saidbacterial species is selected from the group consisting of Streptococcusuberis, Streptococcus agalactiae and Streptococcus pyogenes, and furtherwherein at least one of said CAMP factor epitopes is from the CAMPfactor N-terminal variable region corresponding to the realon defined byamino acids 1-90 of FIG. 2 (SEQ ID NO:2).
 2. The immunogenic polypeptideof claim 1, wherein said CAMP factor epitopes are separated by a spacerconsisting of 1-20 amino acids.
 3. The immunogenic polypeptide of claim1, wherein said CAMP factor epitope from the N-terminal variable regionis interposed within a CAMP factor protein having at least 90% sequenceidentity to the CAMP factor protein depicted at positions 31-258 of FIG.2 (SEQ ID NO:2) or positions 30-255 of FIG. 3 (SEQ ID NO:4), and furtherwherein said CAMP factor protein is from a different streptococcalspecies than the CAMP factor epitope from the N-terminal variableregion.
 4. The immunogenic polypeptide of claim 3, wherein said CAMPfactor epitope from the N-terminal variable region comprises a sequenceof amino acids having at least 90% sequence identity to the contiguoussequence of amino acids depicted at positions 31-87 of FIG. 3 (SEQ IDNO:4) and said CAMP factor protein has at least 90% sequence identity tothe CAMP factor protein depicted at positions 31-258 of FIG. 2 (SEQ IDNO:2).
 5. The immunogenic polypeptide of claim 4, wherein saidpolypeptide comprises a sequence of amino acids having at least 90%sequence identity to the contiguous sequence of amino acids depicted atpositions 27-314 of FIG. 8 (SEQ ID NO:9).
 6. The immunogenic polypeptideof claim 5, wherein said polypeptide comprises the amino acid sequencedepicted at positions 27-314 of FIG. 8 (SEQ ID NO:9).
 7. The immunogenicpolypeptide of claim 6, further comprising a signal sequence.
 8. Theimmunogenic polypeptide of claim 7, wherein said signal sequencecomprises the sequence of amino acids depicted at positions 1-26 of FIG.8 (SEQ ID NO:9).
 9. The immunogenic polypeptide of claim 1, comprisingthe amino acid sequence depicted in FIG. 8 (SEQ ID NO:9).
 10. Animmunogenic composition comprising the immunogenic polypeptide of claim1 and a pharmaceutically acceptable vehicle.
 11. An immunogeniccomposition comprising the immunogenic polypeptide of claim 2 and apharmaceutically acceptable vehicle.
 12. An immunogenic compositioncomprising the immunogenic polypeptide of claim 3 and a pharmaceuticallyacceptable vehicle.
 13. An immunogenic composition comprising theimmunogenic polypeptide of claim 4 and a pharmaceutically acceptablevehicle.
 14. An immunogenic composition comprising the immunogenicpolypeptide of claim 9 and a pharmaceutically acceptable vehicle.
 15. Amethod of producing an immunogenic composition comprising the steps of(1) providing the immunogenic polypeptide of claim 1; and (2) combiningsaid polypeptide with a pharmaceutically acceptable vehicle.